Neovaginal Human Papilloma Virus-Related Squamous Cell Carcinoma in a Transgender Woman.

This case series discusses a human papilloma virus (HPV)­related neovaginal squamous cell carcinoma in a transgender woman and the need for formal gynecologic screening recommendations.

Trends in ovarian, fallopian tube, and primary peritoneal cancer incidence, mortality, and survival: A 15-year population-based analysis.

OBJECTIVE: To characterize trends in ovarian, fallopian tube, and primary peritoneal cancer incidence and incidence-based mortality based on histology and site of origin. METHODS: We obtained age-adjusted incidence and incidence-based mortality for patients with ovarian, fallopian tube, and primary peritoneal cancer from 2000 to 2019 from the US SEER 17 database. Joinpoint 4.9.1.0 was used to characterize log-linear time trends. RESULTS: The incidence and incidence-based mortality of all cancers trended down during the study period. The incidence of epithelial cancers decreased from 2004 to 2019 (AAPC -1.2%, p < 0.001), including that of high-grade (2006-2019: APC -1.2%, p < 0.05) and low-grade (2003-2019: APC -2.4%, p < 0.05) epithelial cancers. There was no change in incidence or incidence-based mortality for ovarian stromal and germ cell cancers. CONCLUSION: There has been a decrease in the incidence and incidence-based mortality of ovarian, fallopian tube, and primary peritoneal cancer, primarily due to reductions in advanced stage epithelial cancers originating in the ovary, fallopian tube, or peritoneum.

Nuclear actin filaments - a historical perspective.

The view on nuclear filaments formed by non-skeletal ß-actin has significantly changed over the decades. Initially, filamentous actin was observed in amphibian oocyte nuclei and only under specific cell stress conditions in mammalian cell nuclei. Improved labeling and imaging technologies have permitted insights into a transient but microscopically apparent filament network that is relevant for chromatin organization, biomechanics of the mammalian cell nucleus, gene expression, and DNA damage repair. Here, we will provide a historical perspective on the developing insight into nuclear actin filaments.

Aberrant nuclear ß-catenin distribution does not prognosticate recurrences of endometrioid endometrial cancers - A retrospective single-institutional study.

OBJECTIVE: Aberrant ß-catenin distribution has been theorized as a predictive biomarker for recurrence in early stage, low grade endometrioid endometrial cancer. METHODS: This retrospective single-institution cohort study reviewed 410 patients with endometrial cancer from May 2018 to May 2022. Only endometrioid histology was included. Demographic and clinicopathological data were collected from the medical records. Univariate and multivariate logistic regressions, and sensitivity analyses for early stage, low grade and no specific molecular profile (NSMP) tumors were performed. RESULTS: 297 patients were included for analysis. Most patients were over 60 years old, White, and with a BMI >30 and early stage low grade disease. Aberrant ß-catenin distribution was found in 135 patients (45.5%) and wild type membranous ß-catenin distribution in 162 (54.5%). While TP53 mutation correlated with endometrial cancer recurrence in this cohort (OR = 4.78), aberrant ß-catenin distribution did not correlate in the overall population (OR = 0.75), the early stage low grade cancers (OR = 0.84), or the NSMP group (OR = 1.41) on univariate or multivariate analysis. No correlation between ß-catenin distribution and local (OR = 0.61) or distant recurrences (OR = 0.90) was detected. CONCLUSIONS: Aberrant ß-catenin distribution did not significantly correlate with recurrence in endometrioid endometrial cancer, nor in the early stage, low grade and NSMP sub-cohorts.

SMARCA4 loss irrelevant for ARID1A mutated ovarian clear cell carcinoma: A case report.

Clear cell carcinomas are rare and relatively chemo-insensitive ovarian cancers with a characteristic molecular pathogenesis. Alterations in ARID1A, a component of the multiprotein chromatin remodeling complex SWI/SNF, are likely early events in the development of ovarian clear cancers arising from atypical endometriosis. Insight into additional driver events and particularly mutations in the same chromatin remodeling complex is limited. Isolated loss of SMARCA4, encoding the ATPase of the SWI/SNF complex, characterizes other aggressive gynecologic cancers including small cell carcinomas of the ovary hypercalcemic type (SCCOHT), undifferentiated endometrial carcinomas (UDEC), and uterine sarcomas (SDUS). The ovarian clear cell carcinoma of a 48-year-old showed in the initial surgical specimen a subclonal loss of SMARCA4 in addition to an ARID1A mutation, i.e., two alterations in the SWI/SNF heterochromatin remodeling complex. We anticipated that the SMARCA4 loss would worsen the disease course in analogy to SCCOHT, UDEC, and SDUS. However, the disease did not accelerate. Instead, the recurrent disease showed restored SMARCA4 expression while retaining the ARID1A mutation. Combinatorial redundancy, diversity and sequence in the SWI/SNF complex assembly as well as DNA- and tissue-specificity may explain the observed irrelevance of SMARCA4 loss in the presented ARID1A mutated ovarian clear cell carcinoma.

Risk of endometrial polyp and surgical intervention in postmenopausal women with proliferative endometrium.

OBJECTIVE: To study the long-term risks of postmenopausal women with proliferative endometrium developing benign uterine pathologies (endometrial polyps and uterine fibroids) and requiring future gynecological interventions, and to compare them with women with atrophic endometrium. DESIGN: Retrospective cohort study of all women aged 55 or over who underwent endometrial biopsy between 1/1997 and 12/2008. Outcome data were available through to 2/2018. Women with proliferative endometrium were compared with those with atrophic endometrium for the presence of endometrial polyps, uterine fibroids, future endometrial biopsy for recurrent vaginal bleeding, and future hysteroscopy or hysterectomy. Logistic regression models were used to evaluate the association of endometrial histology and other covariates with the risk of morbidities. MAIN FINDINGS: Postmenopausal women with proliferative endometrium are at higher risk of developing endometrial polyps, uterine fibroids and need for surgical intervention. Of 1808 women who underwent endometrial biopsy during the study period, 962 met inclusion criteria: 278 had proliferative and 684 had atrophic endometrium. Length of surveillance was similar in the two groups (11.9 vs. 11.5 years, p = 0.2). Compared with women with atrophic endometrium, women with proliferative endometrium had significantly higher rates of endometrial polyps (17.3 % vs 9.7 % p = 0.001). Multivariable logistic regression confirmed that women with proliferative endometrium had more fibroids on ultrasound (62.1 % vs 50.3 % 3 = 0.02), and had increased risks of developing endometrial polyps (aOR 1.9, 95 % CI 1.28-3.07, p = 0.002), repeat endometrial biopsy (34.9 % vs. 16.8%p < 0.001) and future hysterectomy or hysteroscopy (26.6 % vs 16.2 % p < 0.001). CONCLUSIONS: In addition to the long-term increased risk of cancer, postmenopausal women with proliferative endometrium are more likely to have future bleeding, surgical interventions and diagnosis of endometrial polyps. Medical management to reduce estrogenic activity and associated risks may be considered in these cases.

Does lymph node assessment change the prognostic significance of substantial LVSI and p53 status in endometrial endometrioid carcinoma?

OBJECTIVE: The PORTEC-2 update suggested that substantial lymphovascular space invasion (LVSI) and abnormal p53 expression (p53abnl) predict for poorer outcomes and that these patients should be treated with external beam radiation therapy (EBRT). We aim to determine if patients with these risk factors who undergo a lymph node (LN) assessment show similar outcomes. METHODS: We retrospectively reviewed 126 patients with FIGO 2009 stage IA grade 3, stage IB grade 1-2, and stage IIIC (positive LN but no other stage II/III risk factors) endometrioid endometrial cancer who underwent LN assessment. Local (LR), regional recurrences (RR), and distant metastases were analyzed using competing risk methods, and overall survival (OS) was analyzed using Kaplan-Meier. RESULTS: Median follow-up time was 37.2 months. OS was significantly different between patients with and without p53abnl expression (16.7% versus 3.1% deceased), and between patients with and without LVSI (11.1% versus 1.5% deceased; p < 0.01 for both). The 2-year cumulative incidence of LR for patients with p53abnl versus wild type p53 and LVSI versus no LVSI was 11.1% (95% CI 0-25.6) versus 2.2% (95% CI 0-5.25; p = 0.04), and 11.4% (95% CI 2.0-20.9) versus 0%, respectively (p < 0.01). The 2-year cumulative RR in patients with LVSI versus no LVSI was 6.9% (95% CI 0-14.4) versus 0% (p = 0.05). No patients who completed pelvic RT experienced an in-field recurrence. CONCLUSIONS: Despite LN assessment, patients with high-intermediate risk early-stage or stage IIIC (with positive lymph nodes only but no other stage II or III risk factors) endometrial cancer with p53abnl expression and/or LVSI have worse outcomes. These patients may derive benefit from intensification with EBRT to improve local and pelvic control.

Adjuvant radiation therapy in early-stage endometrial cancer with abnormal beta-catenin expression is associated with improved local control.

OBJECTIVES: Emerging data suggests that abnormal (nuclear) ß-catenin expression in some settings is associated with poorer outcomes. Our study aimed to verify the significance of abnormal ß-catenin expression in early-stage endometrial cancer patients and determine if adjuvant radiation therapy (RT) improves local control. METHODS: We identified 213 patients with FIGO 2018 stage I-II endometrioid endometrial cancer who underwent surgery from 2009 to 2021 with ß-catenin expression assessed. Vaginal, regional, and distant recurrences were analyzed using competing risk methods, and overall survival was analyzed using Kaplan-Meier. RESULTS: Median follow up was 53.2 months; 6.9% experienced vaginal, 8.2% regional, and 7.4% distant recurrence. For the entire cohort, abnormal ß-catenin expression was significantly associated with vaginal recurrence and remained significant on multivariate analysis (p = 0.03). There were 114 patients in the no specific molecular profile (NSMP) subgroup, and abnormal ß-catenin expression was present in 46.5%. In the NSMP subgroup, abnormal ß-catenin expression was associated with increased rates of vaginal recurrence (p = 0.06). Abnormal ß-catenin expression in the NSMP subgroup was significant on multivariate analysis for vaginal recurrence (p = 0.04). RT significantly decreased vaginal recurrences in the entire cohort in patients with abnormal ß-catenin expression (0%) versus wild type expression (17.5%; p = 0.03). In the NSMP subgroup 0% of patients who received RT versus 20.9% of patients who did not receive RT experienced a vaginal recurrence (p = 0.03). CONCLUSION: Use of adjuvant RT for stage I-II NSMP endometrial cancer with abnormal ß-catenin expression improved local control. RT should be considered in these patients to decrease risk of vaginal recurrences.

Differential Intracellular Protein Distribution in Cancer and Normal Cells-Beta-Catenin and CapG in Gynecologic Malignancies.

It is well-established that cancer and normal cells can be differentiated based on the altered sequence and expression of specific proteins. There are only a few examples, however, showing that cancer and normal cells can be differentiated based on the altered distribution of proteins within intracellular compartments. Here, we review available data on shifts in the intracellular distribution of two proteins, the membrane associated beta-catenin and the actin-binding protein CapG. Both proteins show altered distributions in cancer cells compared to normal cells. These changes are noted (i) in steady state and thus can be visualized by immunohistochemistry-beta-catenin shifts from the plasma membrane to the cell nucleus in cancer cells; and (ii) in the dynamic distribution that can only be revealed using the tools of quantitative live cell microscopy-CapG shuttles faster into the cell nucleus of cancer cells. Both proteins may play a role as prognosticators in gynecologic malignancies: beta-catenin in endometrial cancer and CapG in breast and ovarian cancer. Thus, both proteins may serve as examples of altered intracellular protein distribution in cancer and normal cells.

Technetium Tc 99m tilmanocept fails to detect sentinel lymph nodes in endometrial cancer.

Background: Technetium Tc 99m tilmanocept is a synthetic radiotracer specifically designed for sentinel lymph node (SLN) mapping that has been FDA-approved in breast cancer, melanoma, and head and neck cancer. No published studies exist for the use of this radiotracer in endometrial cancer. Objective: The primary objective was to determine the detection rate of bilateral SLNs in endometrial cancer with the concurrent use of technetium Tc 99m tilmanocept and ICG. Methods: An open-label, single cohort, prospective feasibility study was conducted with participants receiving preoperative cervical injections of technetium Tc 99m tilmanocept followed by subsequent imaging and SPECT/CT. Intraoperative ICG injections were administered for all patients with near-infrared imaging used to visualize lymphatic vessels and nodes. A laparoscopic gamma counter was used to detect radioactive SLN intraoperatively. Results: All six evaluated patients had FIGO grade 1 or 2 endometrioid histology. Stage IA/IB were in 33% and 66% of patients, respectively. Tilmanocept did not map any SLN in the first six patients but instead showed retention of the tracer in the cervical stroma, leading to study discontinuation for futility. ICG mapped bilateral SLN in all patients with the most common location being the external iliac region, followed by the obturator and common iliac areas. All patients had CD206 positive staining throughout the full wall thickness of ectocervix, transformation zone, endocervix, and lymphatic vessels. No patients experienced adverse events. Conclusion: Technetium Tc 99m tilmanocept did not detect SLN in early stage endometrial cancers and is unlikely to improve bilateral detection rate compared to ICG alone. ICG remains a standard technique for SLN detection in low stage, low grade endometrial cancer.

Prospective molecular classification of endometrial carcinomas: institutional implementation, practice, and clinical experience.

The comprehensive genomic analysis of endometrial carcinoma (EC) by The Cancer Genome Atlas (TCGA) led to the discovery of four distinct and prognostically significant molecular subgroups. Molecular classification has the potential to improve risk-stratification when integrated with clinicopathologic features and has recently been included in national and international patient management EC guidelines. Thus, the adoption of molecular classification into routine pathologic and clinical practice is likely to grow significantly in the upcoming years. Establishing an efficient and standardized workflow for performing molecular classification on ECs, and reporting both the molecular and histologic findings in an integrative manner, is imperative. Here we describe our effort to implement rapid and routine molecular classification on all ECs diagnosed at our institution. To this effect, we performed immunohistochemistry as a surrogate marker for identifying genetic and/or epigenetic alterations in DNA mismatch repair (e.g., MLH1, PMS2, MSH6, MSH2), and TP53 genes. In addition, we have developed and employed a single-gene POLE SNaPshot assay, which is a rapid and analytically sensitive method for detecting select POLE exonuclease domain mutations (EDMs). We report our molecular testing workflow and integrative reporting system as well as the clinicopathologic and molecular features of 310 ECs that underwent routine molecular classification at our institution. The 310 ECs were molecularly classified as follows: 15 (5%) POLE mutant (POLEmut), 79 (25%) mismatch repair-deficient (MMRd), 135 (44%) no specific molecular profile (NSMP), and 81 (26%) p53 abnormal (p53abnl). This work provides an initial framework for implementing routine molecular classification of ECs.

Cancer of the ovary, fallopian tube, and peritoneum: 2021 update.

In 2014, FIGO's Committee for Gynecologic Oncology revised the staging of ovarian cancer, incorporating ovarian, fallopian tube, and peritoneal cancer into the same system. Most of these malignancies are high-grade serous carcinomas (HGSC). Stage IC is now divided into three categories: IC1 (surgical spill); IC2 (capsule ruptured before surgery or tumor on ovarian or fallopian tube surface); and IC3 (malignant cells in the ascites or peritoneal washings). The updated staging includes a revision of Stage IIIC based on spread to the retroperitoneal lymph nodes alone without intraperitoneal dissemination. This category is now subdivided into IIIA1(i) (metastasis ≤10 mm in greatest dimension), and IIIA1(ii) (metastasis >10 mm in greatest dimension). Stage IIIA2 is now "microscopic extrapelvic peritoneal involvement with or without positive retroperitoneal lymph node" metastasis. This review summarizes the genetics, surgical management, chemotherapy, and targeted therapies for epithelial cancers, and the treatment of ovarian germ cell and stromal malignancies.

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission.

Förster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

In invasion assays, the breast cancer cell nucleus leads the way.

OBJECTIVE: Cancer cell metastasis determines disease prognosis. During cancer cell metastasis, the cancer cell and the cancer cell nucleus have to undergo extreme shape changes. To monitor shape changes of cancer cells and cancer cell nuclei and the positioning of the cancer cell nucleus during cancer cell invasion, a customized invasion assay with 8-µm pores and reconstituted basal membrane was imaged using fluorescence live-cell microscopy. RESULTS: The observed cells changed their shape from a distinct fibroblast-like spindle shape to an amoeboid shape without polarization immediately after the passage through an 8-µm pore of the invasion assay. During the process of invasion, the cancer cell centered the cancer cell nucleus over the 8-µm pore, and cancer cell nucleus and adjacent cytoplasmic areas moved first through such a pore. Seemingly testing if the largest and least deformable organelle may fit, the cancer cell nucleus led the way through the porous membrane of the invasion assay.

Vulvar sarcoma outcomes by histologic subtype: a Surveillance, Epidemiology, and End Results (SEER) database review.

OBJECTIVE: Vulvar cancers account for 5% of all gynecologic malignancies; only 1%-3% of those vulvar cancers are primary vulvar sarcomas. Given the rarity of vulvar sarcomas, outcome data specific to histopathologic subtypes are sparse. The aim of this study was to identify clinical and pathologic factors of primary vulvar sarcomas that are associated with survival and may inform treatment decisions. METHODS: The Surveillance, Epidemiology, and End Results (SEER) database was searched for women diagnosed with vulvar sarcoma between 1973 and 2018. We identified 315 patients and reviewed their demographic, clinicopathologic, surgical, and survival information. Statistical analyses included χ2 and t-tests, Kaplan-Meier survival, and Cox regression analyses. RESULTS: The most common histopathologies of vulvar sarcomas were dermatofibrosarcomas (85/315, 27%) and leiomyosarcomas (72/315, 22.9%). Rhabdomyosarcomas (18/315, 5.7%), liposarcomas (16/315, 5.1%), and malignant fibrous histiocytomas (16/315, 5.1%) were less frequent. The majority of patients underwent surgery (292/315, 92.7%), which included lymph node dissections in 21.6% (63/292). Survival and lymph node involvement varied significantly with histologic subtype. The 5-year disease-specific survival for dermatofibrosarcomas, liposarcomas, and fibrosarcomas was 100% and only 60.3% and 62.5% for malignant fibrous histiocytomas and rhabdomyosarcomas, respectively. None of the patients with (dermato)fibrosarcomas, liposarcomas, or leiomyosarcomas had positive lymph nodes, in contrast to rhabdomyosarcomas and malignant fibrous histiocytomas with 77.8% and 40% positive lymph nodes, respectively. The 5-year disease-specific survival for women with positive lymph nodes was 0%. CONCLUSIONS: Vulvar sarcomas are heterogeneous with survival highly dependent on the histopathologic subtype. While surgical excision is the mainstay of treatment for all vulvar sarcomas, staging lymphadenectomy should be deferred for (dermato)fibrosarcomas, liposarcomas, and leiomyosarcomas as there were no cases of lymph nodes metastases.

Long-term outcome of postmenopausal women with proliferative endometrium on endometrial sampling.

BACKGROUND: Proliferative endometrium has been reported in 15% of endometrial biopsies of women aged 50 years and older. Contrary to endometrial hyperplasia, proliferative endometrium has not been associated with the risk of endometrial cancer. OBJECTIVE: This study aimed to report on the long-term outcome of postmenopausal women who received a diagnosis of proliferative endometrium. STUDY DESIGN: This is a retrospective cohort study of 1808 women aged 55 years and older who underwent endometrial sampling between January 1997 and December 2008. Outcome data were available through February 2018. Women with a proliferative endometrium were compared with those with an atrophic endometrium for future development of endometrial hyperplasia or cancer. A subanalysis was performed for those who presented with postmenopausal bleeding. Uni- and multivariable logistic regression analyses were used to assess for confounders. RESULTS: In this study, 297 women (16.4%) received a diagnosis of proliferative endometrium. Furthermore, 962 women met the inclusion criteria. Among those women, 278 had a proliferative endometrium, and 684 had an atrophic endometrium. Women with a proliferative endometrium were younger (61.2 vs 64.5 years; P<.0001) and had a higher body mass index (33.9 vs 30.6 kg/m2; P<.0001). More African American women had a proliferative endometrium. Both groups had a similar length of surveillance (11.9 vs 11.5 years; P=.27). Women with a proliferative endometrium had a higher risk of developing endometrial hyperplasia or cancer (11.9% vs 2.9%; P<.0001), any endometrial cancer (5.8% vs 1.8%; P=.002), atypical endometrial hyperplasia (2.2% vs 0.4%; P=.02), and nonatypical endometrial hyperplasia (2.0% vs 0.7%; P=.001). The risk of developing endometrial cancer and endometrial hyperplasia remained similar after excluding cases on hormonal replacement therapy (12.2% vs 3%; P=.001). On logistic regression analysis, proliferative endometrium histology (odds ratio, 3.89; 95% confidence interval, 2.03-7.49; P<.0001), age >60 years (odds ratio, 1.98; 95% confidence interval, 1.03-3.82; P=.04), and body mass index >35 kg/m2 (odds ratio, 2.3; 95% confidence interval, 1.09-4.83; P<.0001) remained significant risk factors for progression to cancer. CONCLUSION: One of the 6 postmenopausal women who underwent endometrial sampling had a proliferative endometrium. Furthermore, 11.9% of women developed endometrial hyperplasia or cancer, a 4-fold greater incidence than women with an atrophic endometrium. The findings of this study suggest that long-term monitoring is warranted for women with postmenopausal bleeding and a proliferative endometrium histology. Further studies are needed to examine if a treatment is required to negate the risk of unopposed estrogen.

Dissecting Oligomeric States with Photoactivated Localization Microscopy: A Numerical Model.

Although photoactivated localization microscopy offers the potential to interrogate protein interactions in the physiological environment of a cell, uncertainties in the detection efficiency of photoactivatable proteins lead to complications with data interpretation. Here, we present a numerical model that provides probabilities to detect neighboring molecules dependent on their oligomerization status, density, detection efficiency, and radius, and can be used to assess oligomeric states or detection efficiencies of two molecular species. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Sentinel Lymph Node Biopsies in Endometrial Cancer: Practice Patterns among Gynecologic Oncologists in the United States.

STUDY OBJECTIVE: To evaluate practice patterns among gynecologic oncologists with regard to sentinel lymph node injection and biopsy in endometrial cancer. DESIGN: An observational study with no control group. SETTING AND PATIENTS: Active members of the Society of Gynecologic Oncology. INTERVENTIONS: After institutional review board approval, we performed an online survey among active members of the Society of Gynecologic Oncology. Members were contacted via e-mail and their answers anonymously captured. Study data were collected using REDCap (REDCap developed by Vanderbilt University, Nashville TN). MEASUREMENTS AND MAIN RESULTS: Three hundred eighteen of 1216 listed members completed the online survey. The majority of respondents (82.7%) perform sentinel lymph node sampling for endometrial cancer staging. Most technical aspects of sentinel lymph node sampling were consistently applied by the vast majority of respondents, including the choice of indocyanine green as a lymphatic tracer (97.3%) and its injection into the cervix (100%). Other technical aspects of sentinel lymph node sampling, such as the depth of injection, varied among respondents. Although 50.9% of the respondents perform an intraoperative assessment of the uterus by frozen section, only 17.9% assess sentinel lymph nodes by frozen section and/or touch prep. Some of the respondents' approaches are based on limited data, including (1) the use of sentinel lymph node injection and biopsy for high-risk histologies (performed by 69%-75% of the respondents dependent on the histology), (2) omitting side-specific completion lymphadenectomy in the absence of sentinel node mapping (in up to 57.8%), or (3) when lymph node metastases are present (in 39.9%). CONCLUSION: In summary, despite the growing use of sentinel lymph node injection and biopsy in endometrial cancer, practice patterns vary considerably among providers sampled by this survey. Some of the decisions are based on limited evidence and, in some instances, deviate from current published guidelines.

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells.

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells and protein ensembles. Thus far, no method has been available to quantify in bulk and in live cells how many of the PA-FPs expressed are photoactivated to fluoresce. Here, we present a protocol involving internal rulers, i.e., genetically coupled spectrally distinct (photoactivatable) fluorescent proteins, to ratiometrically quantify the fraction of all PA-FPs expressed in a cell that are switched on to be fluorescent. Using this protocol, we show that different modes of photoactivation yielded different photoactivation efficiencies. Short high-power photoactivation with a confocal laser scanning microscope (CLSM) resulted in up to four times lower photoactivation efficiency than hundreds of low-level exposures applied by CLSM or a short pulse applied by widefield illumination. While the protocol has been exemplified here for (PA-)GFP and (PA-)Cherry, it can in principle be applied to any spectrally distinct photoactivatable or photoconvertible fluorescent protein pair and any experimental set-up.