Detection of Radioactive DNA in Polyacrylamide Gels.

Bands of radioactive DNA separated by polyacrylamide gel electrophoresis may be detected by autoradiography or phosphorimaging. Analytical polyacrylamide gels containing radioactive DNA are usually fixed and dried before autoradiography. However, if bands of radioactive DNA are to be recovered from the gel, the gel should generally not be fixed or dried.

EXIRAD-3D: Fast automated three-dimensional autoradiography.

INTRODUCTION: Autoradiography is an established technique for high-resolution imaging of radiolabelled molecules in biological tissue slices. Unfortunately, creating a 3D image from a set of these 2D images is extremely time-consuming and error-prone. MicroSPECT systems provide such 3D images but have a low resolution. Here we present EXIRAD-3D, a fast automated method as an alternative for 3D autoradiography from coupes based on ultra-high resolution microSPECT technology. METHODS: EXIRAD-3D uses a very small bore focusing multi-pinhole collimator mounted in a SPECT system with stationary detectors (U-SPECT/CT, MILabs B.V. The Netherlands) using a sample holder with integrated tissue cooling to avoid activity leaking or tissue deformation during the scan. The system performance was experimentally evaluated using various phantoms and tissue samples of animals in vivo injected with technetium-99m and iodine-123. RESULTS: The reconstructed spatial resolution obtained with a Derenzo hot rod phantom was 120 µm (or 1.7 nl). The voxel values of a syringe phantom image appear to be uniform and scale linearly with activity. Uptake in tiny details of the mouse knee joint, thyroid, and kidney could be clearly visualized. CONCLUSION: EXIRAD-3D opens up the possibility for fast and quantitative 3D imaging of radiolabelled molecules at a resolution far better than in vivo microSPECT and saves tremendous amounts of work compared to obtaining 3D data from a set of 2D autoradiographs. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: EXIRAD-3D offers superior image resolution over microSPECT, and it can be a very efficient alternative for autoradiography in pharmaceutical and biological studies.

Mapping 241Am Spatial Distribution Within Anatomical Bone Structures Using Digital Autoradiography.

Digital autoradiography with the ionizing radiation quantum imaging detector is used at the US Transuranium and Uranium Registries for visualizing the microdistribution of alpha particles from Am and quantifying the activity. The radionuclide spatial distribution was investigated within cortical and trabecular regions of bone samples from US Transuranium and Uranium Registries case 0846. Multiple specimens from the humerus proximal end, humerus proximal shaft, and clavicle acromial end were embedded in plastic, and 100-µm-thick sections were taken and imaged using the ionizing radiation quantum imaging detector. The detector images were superimposed on the anatomical structure images to visualize Am distribution in cortical bone, trabecular bone, and trabecular spongiosa. Activity concentration ratios were used to characterize Am distribution within different bone regions. The trabecular-to-cortical bone and trabecular-spongiosa-to-cortical bone activity concentration ratios were quantified in both humerus and clavicle. The ionizing radiation quantum imaging detector results were in agreement with those obtained from radiochemical analysis of the remaining bone specimens. The results were compared with International Commission on Radiological Protection default biokinetic model predictions. Digital autoradiography was proven to be an effective method for microscale heterogeneous distribution studies where traditional counting methods are impractical.

Radiation Imagers for Quantitative, Single-particle Digital Autoradiography of Alpha- and Beta-particle Emitters.

Promising therapies are being developed or are in early-stage clinical trials that employ the use of alpha- and beta-emitting radionuclides to cure hematologic malignancies. However, these targeted radionuclide therapies have not yet met their expected potential for cancer treatment. A primary reason is lack of biodistribution, dosimetry, and dose-response information at cellular levels, which are directly related to optimal targeting, achieving a requisite therapeutic dose, and assessing the safety profile in normal organs and tissues. The current set of imaging tools, such as film autoradiography, scintigraphy, and SPECT/CT, available to researchers and clinicians do not allow the effective assessment of radiation absorbed dose distributions at cellular levels because resolutions are poor, measurement and analytical times are long, and the spatial resolutions are low-generally resulting in poor signal-to-noise ratios. Recently, new radiation digital autoradiography imaging tools have been developed that promise to address these challenges. They include scintillation-, gaseous-, and semiconductor-based radiation-detection technologies that localize the emission location of charged particles on an event-by-event basis at resolutions up to 20 µm FWHM for alpha and beta emitters. These imaging systems allow radionuclide activity concentrations to be quantified to unprecedented levels (mBq/µg) and provide real-time imaging and simultaneous imaging capabilities of both high- and low-activity samples without dynamic range limitations that plague traditional autoradiography. Additionally, large-area imagers are available (>20 × 20 cm2) to accommodate high-throughput imaging studies. This article reviews the various detector classes and their associated performance trade-offs to provide researchers with an overview of the current technologies available for selecting an optimal detector configuration to meet imaging requirement needs.

Bioinformatics Approach to Identify Novel AMPK Targets.

In silico analysis of Big Data is a useful tool to identify putative kinase targets as well as nodes of signaling cascades that are difficult to discover by traditional single molecule experimentation. System approaches that use a multi-tiered investigational methodology have been instrumental in advancing our understanding of cellular mechanisms that result in phenotypic changes. Here, we present a bioinformatics approach to identify AMP-activated protein kinase (AMPK) target proteins on a proteome-wide scale and an in vitro method for preliminary validation of these targets. This approach offers an initial screening for the identification of AMPK targets that can be further validated using mutagenesis and molecular biology techniques.

Technical Note: Scintillation well counters and particle counting digital autoradiography devices can be used to detect activities associated with genomic profiling adequacy of biopsy specimens obtained after a low activity 18 F-FDG injection.

PURPOSE: Genomic profiling of biopsied tissue is the basis for precision cancer therapy. However, biopsied materials may not contain sufficient amounts of tumor deoxyribonucleonic acid needed for the analysis. We propose a method to determine the adequacy of specimens for performing genomic profiling by quantifying their metabolic activity. METHODS: We estimated the average density of tumor cells in biopsy specimens needed to successfully perform genomic analysis following the Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) protocol from the minimum amount of deoxyribonucleonic acid needed and the volume of tissue typically used for analysis. The average 18 F-FDG uptake per cell was assessed by incubating HT-29 adenocarcinoma tumor cells in 18 F-FDG containing solution and then measuring their activity with a scintillation well counter. Consequently, we evaluated the response of two devices around the minimum expected activities which would indicate genomic profiling adequacy of biopsy specimens obtained under 18 F-FDG PET/CT guidance. Surrogate samples obtained using 18G core needle biopsies of gels containing either 18 F-FDG-loaded cells in the expected concentrations or the corresponding activity were measured using autoradiography and a scintillation well counter. Autoradiography was performed using a CCD-based device with real-time image display as well as with digital autoradiography imaging plates following a 30-min off-line protocol for specimen activity determination against previously established calibration. RESULTS: Cell incubation experiments and estimates obtained from quantitative autoradiography of biopsy specimens (QABS) indicate that specimens acquired under 18 F-FDG PET/CT guidance that contained the minimum amount of cells needed for genomic profiling would have an average activity concentration in the range of about 3 to about 9 kBq/mL. When exposed to specimens with similar activity concentration, both a CCD-based autoradiography device and a scintillation well counter produced signals with sufficient signal-to-background ratio for specimen genomic adequacy identification in less than 10 min, which is short enough to allow procedure guidance. CONCLUSION: Scintillation well counter measurements and CCD-based autoradiography have adequate sensitivity to detect the tumor burden needed for genomic profiling during 18 F-FDG PET/CT-guided 18G core needle biopsies of liver adenocarcinoma metastases.

Actinide bioimaging in tissues: Comparison of emulsion and solid track autoradiography techniques with the iQID camera.

This work presents a comparison of three autoradiography techniques for imaging biological samples contaminated with actinides: emulsion-based, plastic-based autoradiography and a quantitative digital technique, the iQID camera, based on the numerical analysis of light from a scintillator screen. In radiation toxicology it has been important to develop means of imaging actinide distribution in tissues as these radionuclides may be heterogeneously distributed within and between tissues after internal contamination. Actinide distribution determines which cells are exposed to alpha radiation and is thus potentially critical for assessing absorbed dose. The comparison was carried out by generating autoradiographs of the same biological samples contaminated with actinides with the three autoradiography techniques. These samples were cell preparations or tissue sections collected from animals contaminated with different physico-chemical forms of actinides. The autoradiograph characteristics and the performances of the techniques were evaluated and discussed mainly in terms of acquisition process, activity distribution patterns, spatial resolution and feasibility of activity quantification. The obtained autoradiographs presented similar actinide distribution at low magnification. Out of the three techniques, emulsion autoradiography is the only one to provide a highly-resolved image of the actinide distribution inherently superimposed on the biological sample. Emulsion autoradiography is hence best interpreted at higher magnifications. However, this technique is destructive for the biological sample. Both emulsion- and plastic-based autoradiography record alpha tracks and thus enabled the differentiation between ionized forms of actinides and oxide particles. This feature can help in the evaluation of decorporation therapy efficacy. The most recent technique, the iQID camera, presents several additional features: real-time imaging, separate imaging of alpha particles and gamma rays, and alpha activity quantification. The comparison of these three autoradiography techniques showed that they are complementary and the choice of the technique depends on the purpose of the imaging experiment.

New simple and low-cost methods for periodic checks of Cyclone® Plus Storage Phosphor System.

The recent large use of the Cyclone® Plus Storage Phosphor System, especially in European countries, as imaging system for quantification of radiochemical purity of radiopharmaceuticals raised the problem of setting the periodic controls as required by European Legislation. We described simple, low-cost methods for Cyclone® Plus quality controls, which can be useful to evaluate the performance measurement of this imaging system.

The origin of in situ hybridization - A personal history.

In situ hybridization is the technique by which specific RNA or DNA molecules are detected in cytological preparations. Basically it involves formation of a hybrid molecule between an endogenous single-stranded RNA or DNA in the cell and a complementary single-stranded RNA or DNA probe. In its original form the probe was labeled with (3)H and the hybrid was detected by autoradiography. The first successful experiments in 1968 involved detection of the highly amplified ribosomal DNA in oocytes of the frog Xenopus, followed soon after by the reiterated "satellite DNA" in mouse and Drosophila chromosomes. Fluorescent probes were developed about ten years later.

Quantitative single-particle digital autoradiography with α-particle emitters for targeted radionuclide therapy using the iQID camera.

PURPOSE: Alpha-emitting radionuclides exhibit a potential advantage for cancer treatments because they release large amounts of ionizing energy over a few cell diameters (50-80 µm), causing localized, irreparable double-strand DNA breaks that lead to cell death. Radioimmunotherapy (RIT) approaches using monoclonal antibodies labeled with α emitters may thus inactivate targeted cells with minimal radiation damage to surrounding tissues. Tools are needed to visualize and quantify the radioactivity distribution and absorbed doses to targeted and nontargeted cells for accurate dosimetry of all treatment regimens utilizing α particles, including RIT and others (e.g., Ra-223), especially for organs and tumors with heterogeneous radionuclide distributions. The aim of this study was to evaluate and characterize a novel single-particle digital autoradiography imager, the ionizing-radiation quantum imaging detector (iQID) camera, for use in α-RIT experiments. METHODS: The iQID camera is a scintillator-based radiation detection system that images and identifies charged-particle and gamma-ray/x-ray emissions spatially and temporally on an event-by-event basis. It employs CCD-CMOS cameras and high-performance computing hardware for real-time imaging and activity quantification of tissue sections, approaching cellular resolutions. In this work, the authors evaluated its characteristics for α-particle imaging, including measurements of intrinsic detector spatial resolutions and background count rates at various detector configurations and quantification of activity distributions. The technique was assessed for quantitative imaging of astatine-211 ((211)At) activity distributions in cryosections of murine and canine tissue samples. RESULTS: The highest spatial resolution was measured at ∼20 µm full width at half maximum and the α-particle background was measured at a rate as low as (2.6 ± 0.5) × 10(-4) cpm/cm(2) (40 mm diameter detector area). Simultaneous imaging of multiple tissue sections was performed using a large-area iQID configuration (ø 11.5 cm). Estimation of the (211)At activity distribution was demonstrated at mBq/µg-levels. CONCLUSIONS: Single-particle digital autoradiography of α emitters has advantages over traditional film-based autoradiographic techniques that use phosphor screens, in terms of spatial resolution, sensitivity, and activity quantification capability. The system features and characterization results presented in this study show that the iQID is a promising technology for microdosimetry, because it provides necessary information for interpreting alpha-RIT outcomes and for predicting the therapeutic efficacy of cell-targeted approaches using α emitters.

Characterization of a double-sided silicon strip detector autoradiography system.

PURPOSE: The most commonly used technology currently used for autoradiography is storage phosphor screens, which has many benefits such as a large field of view but lacks particle-counting detection of the time and energy of each detected radionuclide decay. A number of alternative designs, using either solid state or scintillator detectors, have been developed to address these issues. The aim of this study is to characterize the imaging performance of one such instrument, a double-sided silicon strip detector (DSSD) system for digital autoradiography. A novel aspect of this work is that the instrument, in contrast to previous prototype systems using the same detector type, provides the ability for user accessible imaging with higher throughput. Studies were performed to compare its spatial resolution to that of storage phosphor screens and test the implementation of multiradionuclide ex vivo imaging in a mouse preclinical animal study. METHODS: Detector background counts were determined by measuring a nonradioactive sample slide for 52 h. Energy spectra and detection efficiency were measured for seven commonly used radionuclides under representative conditions for tissue imaging. System dead time was measured by imaging (18)F samples of at least 5 kBq and studying the changes in count rate over time. A line source of (58)Co was manufactured by irradiating a 10 µm nickel wire with fast neutrons in a research reactor. Samples of this wire were imaged in both the DSSD and storage phosphor screen systems and the full width at half maximum (FWHM) measured for the line profiles. Multiradionuclide imaging was employed in a two animal study to examine the intratumoral distribution of a (125)I-labeled monoclonal antibody and a (131)I-labeled engineered fragment (diabody) injected in the same mouse, both targeting carcinoembryonic antigen. RESULTS: Detector background was 1.81 × 10(-6) counts per second per 50 × 50 µm pixel. Energy spectra and detection efficiency were successfully measured for seven radionuclides. The system dead time was measured to be 59 µs, and FWHM for a (58)Co line source was 154 ± 14 µm for the DSSD system and 343 ± 15 µm for the storage phosphor system. Separation of the contributions from (125)I and (131)I was performed on autoradiography images of tumor sections. CONCLUSIONS: This study has shown that a DSSD system can be beneficially applied for digital autoradiography with simultaneous multiradionuclide imaging capability. The system has a low background signal, ability to image both low and high activity samples, and a good energy resolution.

Evaluation of thermal neutron irradiation field using a cyclotron-based neutron source for alpha autoradiography.

It is important to measure the microdistribution of (10)B in a cell to predict the cell-killing effect of new boron compounds in the field of boron neutron capture therapy. Alpha autoradiography has generally been used to detect the microdistribution of (10)B in a cell. Although it has been performed using a reactor-based neutron source, the realization of an accelerator-based thermal neutron irradiation field is anticipated because of its easy installation at any location and stable operation. Therefore, we propose a method using a cyclotron-based epithermal neutron source in combination with a water phantom to produce a thermal neutron irradiation field for alpha autoradiography. This system can supply a uniform thermal neutron field with an intensity of 1.7×10(9) (cm(-2)s(-1)) and an area of 40mm in diameter. In this paper, we give an overview of our proposed system and describe a demonstration test using a mouse liver sample injected with 500mg/kg of boronophenyl-alanine.

Development of a simple and rapid method of precisely identifying the position of 10B atoms in tissue: an improvement in standard alpha autoradiography.

Boron neutron capture therapy (BNCT) can be utilized to selectively kill cancer cells using a boron compound that accumulates only in cancer cells and not in normal cells. Tumor-bearing animals treated by BNCT are routinely used to evaluate long-term antitumor effects of new boron compounds. Alpha-autoradiography is one of the methods employed in the evaluation of antitumor effects. However, a standard alpha-autoradiography cannot detect the microdistribution of (10)B because of the difficulty associated with the superposition of a tissue sample image and etched pits on a track detector with the etching process. In order to observe the microdistribution of (10)B, some special methods of alpha-autoradiography have been developed that make use of a special track detector, or the atomic force microscope combined with X-ray and UV light irradiation. In contrast, we propose, herein, a simple and rapid method of precisely identifying the position of (10)B using the imaging process and the shape of etched pits, such as their circularity, without the need to use special track detectors or a microscope. A brief description of this method and its verification test are presented in this article. We have established a method of detecting the microdistribution of (10)B with submicron deviation between the position of etched pits and the position of reaction in a tissue sample, for a given circularity of etched pits.

Principles of nuclear medicine imaging: planar, SPECT, PET, multi-modality, and autoradiography systems.

The underlying principles of nuclear medicine imaging involve the use of unsealed sources of radioactivity in the form of radiopharmaceuticals. The ionizing radiations that accompany the decay of the administered radioactivity can be quantitatively detected, measured, and imaged in vivo with instruments such as gamma cameras. This paper reviews the design and operating principles, as well as the capabilities and limitations, of instruments used clinically and preclinically for in vivo radionuclide imaging. These include gamma cameras, single-photon emission computed tomography (SPECT) scanners, and positron emission tomography (PET) scanners. The technical basis of autoradiography is reviewed as well.

A proposal for phosphor imager acceptance testing procedure and routine quality controls in nuclear pharmacy practice.

OBJECTIVE: The digital autoradiography system is currently used in nuclear medicine for quantitative imaging of radioactivity distribution (thin layer chromatography samples, tissue sections, and cell cultures). The aim of this study was to define a set of tests for setting up a specific acceptance testing procedure and routine quality controls for this instrument. MATERIALS AND METHODS: Over a 3-month period, we analyzed the active components of the instrument (phosphor screen and photometer) by using suitable self-manufactured equipment (phantoms, lead plate, lead cylinder, and photographic paper) required to realize, in a routine quality program, the following tests: integral uniformity (IU) and differential uniformity (DU) in a useful field of view (UFOV) and a central field of view (CFOV), resolution, geometric linearity, and sensitivity. RESULTS: Screen IU was 19.7 ± 2.3% (UFOV) and 11.1 ± 3.7% (CFOV). Screen DU ranged between 1.6 ± 1.1 and 1.8 ± 0.9% for UFOV and between 1.2 ± 0.4 and 1.4 ± 0.6% for CFOV. Screen resolution measured as full-width at half-maximum was 1.94 ± 0.08 mm. Screen sensitivity was 505.1 ± 10.4 digital light units and ranged between -3.15 and +3.49% with reference to the mean of measured values. Photometer IU was 17.4 ± 0.2% (UFOV) and 13.7 ± 1.1% (CFOV). Photometer DU ranged between 1.9 ± 0.9 and 2.3 ± 1.2% for UFOV and between 1.9 ± 0.8 and 2.1 ± 1.1% for CFOV. Photometer resolution was good (full-width at half-maximum =0.5 ± 0.076 mm). CONCLUSION: Our results suggest that the methodology we propose could be an easy, accurate, quick, and low-cost tool to guarantee the correct instrument basic function.

14C autoradiography with an energy-sensitive silicon pixel detector.

The first performance tests are presented of a carbon-14 ((14)C) beta-particle digital autoradiography system with an energy-sensitive hybrid silicon pixel detector based on the Timepix readout circuit. Timepix was developed by the Medipix2 Collaboration and it is similar to the photon-counting Medipix2 circuit, except for an added time-based synchronization logic which allows derivation of energy information from the time-over-threshold signal. This feature permits direct energy measurements in each pixel of the detector array. Timepix is bump-bonded to a 300 µm thick silicon detector with 256 × 256 pixels of 55 µm pitch. Since an energetic beta-particle could release its kinetic energy in more than one detector pixel as it slows down in the semiconductor detector, an off-line image analysis procedure was adopted in which the single-particle cluster of hit pixels is recognized; its total energy is calculated and the position of interaction on the detector surface is attributed to the centre of the charge cluster. Measurements reported are detector sensitivity, (4.11 ± 0.03) × 10(-3) cps mm(-2) kBq(-1) g, background level, (3.59 ± 0.01) × 10(-5) cps mm(-2), and minimum detectable activity, 0.0077 Bq. The spatial resolution is 76.9 µm full-width at half-maximum. These figures are compared with several digital imaging detectors for (14)C beta-particle digital autoradiography.

The alpha-camera: a quantitative digital autoradiography technique using a charge-coupled device for ex vivo high-resolution bioimaging of alpha-particles.

UNLABELLED: Bioconjugates used in internal radiotherapy exhibit heterogeneous distributions in organs and tumors, implying a risk of nonuniform dose distribution in therapeutic applications using α-particle emitters. Tools are required that provide data on the activity distribution for estimation of absorbed dose on a suborgan level. The α-camera is a quantitative imaging technique developed to detect α-particles in tissues ex vivo. The aim of this study was to evaluate the characteristics of this imaging system and to exemplify its potential use in the development of α-radioimmunotherapy. METHODS: The α-camera combines autoradiography with a scintillating technique and optical registration by a charge-coupled device (CCD). The imaging system characteristics were evaluated by measurements of linearity, uniformity, and spatial resolution. The technique was applied for quantitative imaging of (211)At activity distribution in cryosections of tumors, kidney, and whole body. Intratumoral activity distributions of tumor-specific (211)At-MX35-F(ab')(2) were studied at various times after injection. The postinjection activity distributions in the renal cortex and whole kidneys were compared for (211)At-F(ab')(2) and (211)At-IgG trastuzumab. RESULTS: Quantitative analysis of α-camera images demonstrated that the pixel intensity increased linearly with activity in the imaged specimen. The spatial resolution was 35 ± 11 µm (mean ± SD) and the uniformity better than 2%. Kidney cryosections revealed a higher cortex-to-whole kidney ratio for (211)At-F(ab')(2) than for (211)At-IgG (1.38 ± 0.03 and 0.77 ± 0.04, respectively) at 2 h after injection. Nonuniform intratumoral activity distributions were found for tumor-specific (211)At-MX35-F(ab')(2) at 10 min and 7 h after injection; after 21 h, the distribution was more uniform. CONCLUSION: The characteristics of the α-camera are promising, suggesting that this bioimaging system can assist the development, evaluation, and refinement of future targeted radiotherapy approaches using α-emitters. The α-camera provides quantitative data on the activity distribution in tissues on a near-cellular scale and can therefore be used for small-scale dosimetry, improving the prediction of biologic outcomes with α-particles with short path length and high linear energy transfer.

The spatial resolution of silicon-based electron detectors in beta-autoradiography.

Thin tissue autoradiography is an imaging modality where ex-vivo tissue sections are placed in direct contact with autoradiographic film. These tissue sections contain a radiolabelled ligand bound to a specific biomolecule under study. This radioligand emits beta - or beta+ particles ionizing silver halide crystals in the film. High spatial resolution autoradiograms are obtained using low energy radioisotopes, such as (3)H where an intrinsic 0.1-1 microm spatial resolution can be achieved. Several digital alternatives have been presented over the past few years to replace conventional film but their spatial resolution has yet to equal film, although silicon-based imaging technologies have demonstrated higher sensitivity compared to conventional film. It will be shown in this work how pixel size is a critical parameter for achieving high spatial resolution for low energy uncollimated beta imaging. In this work we also examine the confounding factors impeding silicon-based technologies with respect to spatial resolution. The study considers charge diffusion in silicon and detector noise, and this is applied to a range of radioisotopes typically used in autoradiography. Finally an optimal detector geometry to obtain the best possible spatial resolution for a specific technology and a specific radioisotope is suggested.

Macromolecular synthesis in the livers of aging mice as revealed by electron microscopic radioautography.

For the purpose of studying the aging changes of macromolecular synthesis in animal cells, we studied many groups of aging mice during development and aging from fetal day 19 to postnatal newborn, juvenile, young adults, aged and senescent adults up to 12 and month 24 (2 years). They were injected with (3)H-thymidine, (3)H-uridine or (3)H-leucine, precursors for DNA, RNA and proteins, as well as (3)H-glucose, (3)H-glucosamine, (35)S-sulfuric acid, or (3)H-glycerol for glucide and lipid precursors, respectively, then sacrificed and the liver tissues were taken out, fixed and processed for light and electron microscopic radioautography. On many radioautograms the localization of silver grains demonstrating DNA, RNA and proteins in hepatocytes in respective aging groups were analyzed qualitatively. The number of silver grains and the number of cell organelles in each cell of each animal in respective aging groups were analyzed quantitatively in relation to the aging of individual animals. The results revealed that the localization of respective precursors as well as the number of silver grains in cell nuclei, cell organelles, changed with the aging of animals. The numbers of labeled nuclei and cell organelles, as well as the numbers of silver grains in nuclei and cell organelles changed due to aging of individual animals. The number of mitochondria, the number of labeled mitochondria and the mitochondrial labeling index labeled with silver grains were counted in each hepatocyte. It was demonstrated that the numbers of mitochondria, the numbers of labeled mitochondria and the labeling indices showing DNA, RNA and protein synthesis at various ages from embryonic day 19 to postnatal newborn day 1, 3, 9, 14, adult month 1, 2 and 6, reaching the maxima, then decreased to senile year 1 to 2, indicating the aging changes. The results indicated that mitochondria in hepatocytes synthesized nucleic acids and proteins independently from the nuclei, but their synthetic activities were affected from the aging of the individual animals.