Investigation of Mohs Surgical Margins Using Two Photon Microscopy
Phase: NA
Conditions: Basal Cell Carcinoma
Sponsor: University of Rochester

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Title: Investigation of Mohs surgical margins using two photon fluorescence microscopy
Principal Investigator: Giacomelli, Michael 
1. Purpose of the Study:
In a previous protocol (STUDY00003085, PI: Giacomelli) we performed preliminary two photon 
fluorescence microscopy (TPFM) imaging of surgical excisions and tissue biopsy specimens at both 
URMC and Rochester Dermatologic Surgery (RDS), an outpatient clinic which has a reliance 
agreement under which the RSRB provides IRB supervision. In another ongoing study at RDS 
(STUDY00006388, PI: Giacomelli), we are imaging diagnostic biopsies from patients with 
suspected skin cancer to provide immediate assessment of cancer status. Collectively we have 
imaged several hundred skin cancer and biopsy specimens, demonstrating that TPFM can be used to 
image and detect skin cancer in a dermatological surgery clinic and that TPFM can be performed on 
diagnostic tissues.  
This new study proposes to extend the previous line of research to skin cancer detection on surgical 
margins during Mohs surgery. At present, patients undergoing Mohs surgery for basal cell 
carcinoma (the most common skin cancer) undergo excisions followed by lengthy delays while 
tissue
 is cryosectioned, stained and then evaluated to guide subsequent excision. This study proposes to 
evaluate margins immediately after excision during Mohs surgery using TPFM, potentially saving 
an hour or more of treatment time per patient.  
2. Background:
Mohs surgery.  Basal cell carcinomas (BCC) of the skin are treated each year than all other forms 
of cancer combined. Although slow growing and seldom fatal, they typically occur on sun-exposed 
skin such as the face where treatment can be difficult and costly. Mohs surgery, where the tumor is 
removed in stages using frozen section analysis (FSA) to evaluate each excision before performing 
the next obtains the highest cure rates and minimizes resection of healthy tissue. However, Mohs 
surgery is expensive and labor-intensive because each excision must be cryosectioned for analysis 
while the patient waits with an open surgical wound before the next round of excision or closure of 
surgical wounds can occur. The lengthy delays during Mohs surgery have motivated the search for 
faster, more efficient means to evaluate margins. 
 
Two Photon Fluorescence Microscopy (TPFM).  TPFM is a nondestructive optical microscopy 
technique that is widely used in the life sciences for imaging of live tissue. In previous research 
conducted at BIDMC in Boston, we conducted studies to develop TPFM imaging protocols for skin, 
breast, and prostate cancer.  In reference [1] we developed a system and protocol for imaging breast 
and other surgical specimens. In reference [2] we validated the use of this protocol to image breast 
tissue and verified that fluorescent imaging had no effect on conventional histology, IHC or FISH 
assays.  In reference [3], we performed preliminary investigations of Mohs surgical specimens 
showing that fluorescent imaging may be potentially valuable for guiding surgery. In reference [4], 
we performed more extensive imaging of excised skin cancer specimens, and further demonstrated 

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that two photon imaging did not interfere with subsequent histology or immunohistochemistry in 
dermatologic specimens. Our findings are consistent with that of other groups investigating 
fluorescent histology as a means of evaluating surgical pathology [5].  Finally, concurrent with 
previous work we conducted a clinical study imaging diagnostic prostate specimens from 40 
patients using the two photon imaging protocol proposed here, demonstrating that diagnosis was 
similar on rapid two photon imaging performed immediately after excision as compared to 
subsequent histology of the same specimens [6]. 
In a large study of 127 discarded excisions left over after Mohs surgery, two Mohs surgeons not 
involved in data collection obtained very high agreement with frozen sections when evaluating 
TPFM images (preliminary manuscript attached).  In total, there were 14 disagreements due to tissue 
co-registration and sample preparation which do not reflect true diagnostic accuracy and 8 
specimens (7%) where 1 reviewer diagnosed TPFM differently than frozen sections and one 
reviewer agreed with frozen sections. There were zero co-registered samples were both 
reviewers were incorrect. Thus, TPFM was highly reliable for the diagnosis of BCC on Mohs 
margins.
Previous studies under supervision of the RSRB:  Under STUDY00003085, we imaged 21 
biopsy specimens from Mohs surgery patients that were not required for diagnosis using TPFM and 
demonstrated that BCC could be readily visualized.  These results were published recently in JAMA 
Dermatology [7].  Under STUDY00006388 we are routinely imaging diagnostic biopsy specimens 
from patients prior to Mohs surgery.  While this study is still running, we have not encountered 
problems imaging BCC and have had no adverse events due to TPFM imaging of diagnostic tissues 
at RDS.  
Rochester Dermatologic Surgery (RDS).  RDS is an outpatient dermatologic surgery clinic 
located in Victor NY and owned by Dr. Sherrif Ibrahim, co-investigator on this protocol.  Dr. 
Ibrahim splits his surgical practice between URMC and RDS.  To facilitate collaboration between 
the lab of Michael Giacomelli, located at URMC and Dr. Ibrahim’s practice at both locations, the 
RSRB typically acts as the IRB for studies jointly between URMC and RDS.  In this case, we 
request a reliance agreement. 
3. Administrative Organization
The research location is Rochester Dermatologic Surgery in Victor, NY.  
4. Study Design
4.1. Study Population and Recruitment:
This study proposes to image surgical margins from patients undergoing Mohs surgery at RDS. 
Mohs surgery is an outpatient procedure conducted in a single visit with only local anesthesia. 
During the visit, Dr. Ibrahim or another physician under his supervision will explain to patients 
being treated for basal cell carcinoma (BCC) that they are eligible to participate in a clinical study 
and provide them with a consent form that contains additional information. If interested, he or 
another study member will obtain consent (see Consent procedure and documentation below).  

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The study population will be adult patients undergoing Mohs surgery at RDS. This study will be 
powered to reject the null hypothesis that the true sensitivity and specificity of TPFM for the 
evaluation BCC margins is less than 85% at a confidence level of 95%. If the true sensitivity and 
specificity are each 95%, and 38% of patients have a positive surgical margin, then 123 patients 
would be required. Furthermore, to account for screen fails and patients for which all data is not 
available during analysis, we request an additional 12 patients Thus, we anticipate an enrollment of 
135 patients.
4.2. Study Activities:
This study is an interventional study of skin cancer that will use TPFM to evaluate surgical margins 
followed by standard of care FSA for confirmation. This study will image routine skin cancer 
margins from the practice of Dr. Ibrahim at RDS and image them using TPFM to determine if 
TPFM can accurately detect BCC on surgical margins. To protect patients, following TPFM 
imaging, patients will receive standard of care evaluation using cryosectioned H&E slides, thus this 
design will ensure that all patients will be adequately treated and that TPFM cannot deny any 
patient complete treatment.  
Under Dr. Ibrahim or another physician under his supervision’s direction, individual excisions from 
consenting patients will be selected, fluorescently labeled (protocol described below, already 
approved in STUDY00006388) and diagnosed using the two photon microscope at RDS. A full image 
of the tissue will also be digitally recorded. Following imaging, specimens will be cryosectioned as per 
normal procedure. In the event of definitive BCC on margins, Dr. Ibrahim or another physician under 
his supervision may choose to immediately excise the specific area of BCC rather than wait the 
additional 30-60 minutes for FSA.  If no BCC is found, or if TPFM images are equivocal, the FSA 
images will be consulted first. As a result of this design, the last excision (which must always be 
negative or else surgery is continued) will always be confirmed on FSA, thus ensuring standard 
treatment.
In STUDY00003085, we deployed and tested a compact two photon microscope engineered for use in 
a dermatologic clinic at RDS. In testing, this device could rapidly perform 3D imaging of entire Mohs 
margin specimens in approximately 10 minutes or less. In normal practice, specimens may wait 20 
minutes or more in queue for cryosectioning or post cryosectioning evaluation, so the delay for study 
activities will fit into existing delays built into clinical workflows.  Thus, TPFM imaging will not 
significantly prolong surgery. 
Following conclusion of surgery there will be no further study-related contact with patients. 
Following treatment, the results of the TPFM evaluation of each Mohs excisions will be compared to 
the results from FSA to assess the overall accuracy of TPFM. To perform this comparison, we will 
record two photon fluorescence images of each surgical margin as well as a white light image for 
orientation.  We will further record images of FSA histology in the event that TPFM images disagree 
with FSA for comparison.
Information Retained

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This study will record basic demographic information, microscopic images recorded from TPFM and 
FSA, and the results of diagnostic procedures performed using FSA (for example, the presence of 
cancer on the surgical margin).
Each patient’s name, RDS case number and basic demographic information (age, race, sex and surgery 
location) will be recorded in URMC’s RedCap. To enable deidentification of image data, each patient 
will be assigned an index number that will be stored in RedCap.  The diagnosis based on TPFM and 
conventional microscopic images will also be recorded under the same deidentified index number. 
Signed consent forms with the patient’s name and index number will be stored in locked cabinets at 
RDS. Following completion of the study, consent forms will be stored for 3 years and will then be 
destroyed. In general, we expect that no medical record review will be required and patients will be 
able to state their age, race and surgery location during the consent process.  However, we will record 
the RDS case number (which can be used by RDS staff to look up specific procedures) in the event that 
information (e.g. FSA slide images) must be retrieved on a later date. Thus, we are also requesting 
permission to retrieve demographic, procedure information (e.g. location) or slide images from RDS 
medical records using the case number.  
Access to information
Access to the RDS case number in RedCap (the only PHI recorded electronically) will be restricted to 
personnel assisting with data collection and consenting using RedCap record permissions.  Study 
personnel will not directly access RDS computers or records, and will instead request that Dr. Ibrahim 
or one of his clinic staff provide information if a medical record must be reviewed.  In this way, Dr. 
Ibrahim will ensure that case numbers are translated into other information only as required.  Signed 
consent forms (the only place patient names are recorded) will be locked by RDS staff, and only study 
personnel involved in consenting patients will have access to consent forms. Thus, Dr. Ibrahim and his 
staff, who already have access to patient information as part of their professional duties, will serve as 
gate keepers and ensure that access to patient names and medical records are performed only as 
approved in this protocol. 
Individual TPFM or FSA images will be stored at URMC on an offline data server in the Giacomelli 
lab (URMC 5-8145).  Images will be stored without PHI and will be identified only by the index 
number. Thus, all PHI will remain in RedCap or at RDS where patient records would anyway be 
stored.  Following completion of the study, deidentified TPFM and FSA images will be stored 
indefinitely and will shared with the scientific community consistent with NIH policies on data sharing.
Diagnostic Tissue Staining Protocol and Noninterference With Histology
TPFM is a nondestructive optical imaging technique that can be performed on fresh tissue without 
fixation, embedding or microtome cutting. This imaging protocol uses dilute fluorescent dyes to 
label tissue analogously to hematoxylin and eosin staining in conventional H&E histopathology. 
Fluorescent dyes will be applied to the tissue specimen ex vivo hence the fluorescent dyes will never 
come in contact with the patient and do not pose a direct risk to patient health. 
Unlike conventional histopathology in which stains are chosen that are visible to the human eye, 
fluorescent microscopy uses fluorescent dyes that have much weaker absorption and are not visible 

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the human eye on histology slides. Our protocol performs staining with a solution containing one of 
the reagents acridine orange (AO) at less than 1 millimolar (~20x lower concentration than typical 
histology stains), or SYBR Green (SG) at less than 100 micromolar (~200x lower concentration) 
and sulforhodamine 101 (SR101) at less than 1 millimolar concentration (~20x lower concentration 
than typical histology stains), for up to 3 minutes treatment dissolved in 70-100% ethanol. AO is a 
fluorescent stain occasionally used in blood smears to identify infectious organisms. SG is a newer 
DNA label that has improved specificity, but otherwise performs similar functions and fluoresces at 
identical wavelengths to AO.  SR101 is a dye that is chemically related to eosin that stains stroma 
and fluoresces red. These stains provide nuclear and stromal fluorescent contrast analogous to 
hematoxylin and eosin staining in conventional histopathology. None of these agents are routinely 
used to label skin tissue during routine histopathology and therefore represent a change in 
procedure. However, because neither agent is visible to the human eye on histology slides at the low 
concentration used, labeling is not apparent on conventional visible light microscopy. Furthermore, 
all agents only temporarily label human tissue, and are removed from tissue by routine 
histological processing solvents such as ethanol as used during paraffinization or staining 
during cryosectioning [4]. Simple soaking in standard histological processing solvents (ethanol or 
xylene) extracts all labels. During testing with fresh tissue conducted in the Department of 
Pathology at URMC, all reagents were removed to undetectable levels during standard 
paraffinization and prior to conventional staining on a commercial tissue processor. In testing at 
RDS, FSA performed on TPFM samples was visually identical to unstained samples.
In addition to verifying that the regents are extracted by routine histological processing, we have 
further conducted testing to demonstrate noninterference with IHC. Specimens were stained at the 
concentrations used in this protocol with AO/SR101 and then submitted for routine IHC (ER, PR, 
MART1, HER2). SG was stained at concentrations used in this protocol and submitted for routine 
IHC (MART1, BEREP4, and pancytokeratin as would be used for dermatology specimens). No 
difference was observed, and no fluorescence was visible. These results are consistent with our 
finding that all three agents are removed during paraffinization and not physically present 
after paraffinization. 
Investigational Device Exemption not required: The TPFM system is exempt from the IDE 
requirements Per 21 CFR 812.2 because it is noninvasive, does not require an invasive sampling 
procedure, does not introduce energy into subjects, and will not be used as a diagnostic without 
confirmation from an established diagnostic.  Therefore, no IDE is provided.
5. Inclusion and Exclusion Criteria
Patients 18 years or older being treated for basal cell carcinoma of the skin with Mohs surgery at 
Rochester Dermatologic Surgery will be eligible to participate.  As RDS is a small clinic that rarely 
treats non-English speaking patients, translation services are not typically available.  Therefore, as 
we cannot ensure informed consent in this case, patients that are unable to read an English-language 
consent form will be excluded from the study.  
6. Recruitment Methods

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Dr. Ibrahim or another staff member under his supervision will inform patients meeting the inclusion 
criteria that they are eligible to participate in a clinical study while discussing treatment with them prior 
to surgery.  
7. Informed Consent:
We will use a written consent form to document informed consent. The consent form is attached to 
this protocol submission.  Signed consent forms will be stored on file at RDS. 
Consent procedure and documentation
The patient will be informed that images will be acquired of their surgical excision and that these 
images may be used to guide treatment. The patient will be informed that regardless of choosing to 
participate in the study, final treatment status will be determined according to standard of care.  Finally, 
a consent form will be provided to the patient that includes information summarizing the study. If the 
patient agrees to participate, Dr. Ibrahim or someone under his supervision will answer any questions, 
record their signature, and record their demographic information.  Consenting will be performed shortly 
before the patient’s surgery.  
8. Risks and Benefits:
Risks from TPFM imaging. This imaging protocol involves staining freshly excised pathology 
specimens with dilute solutions of fluorescent contrast agents.  These agents are not routinely used 
and differ from the standard postoperative histology protocol. However, this is no more than 
minimum risk because (1) the dye concentrations are low compared to standard concentrations used 
for dyes such as hematoxylin and eosin, (2) all dyes are highly soluble in histological processing 
agents (e.g. alcohol and xylene) and can be removed during routine processing (3) Fluorescent label 
concentration decreases exponentially below the tissue surface, and therefore normal sectioning 
removes most or all of the exposed tissue. Furthermore, we have conducted an investigation with the 
Department of Pathology at BIDMC that verifies that no interference is observed on pathology 
specimens (as described in reference 2) and then replicated this investigation with the Department of 
Pathology at URMC and confirmed that all agents are completed removed using their standard 
tissue processing protocol. We have further tested IHC on treated specimens and found no 
interference or residual fluorescent signal. All imaging will be performed at the surgeon’s 
discretion, and while interference or complications from fluorescent imaging have never been 
observed, if any are suspected, imaging can be halted, and the fluorescent labels washed out with 
ethanol (a standard reagent used during staining of cryosectioned tissues) or simply sectioned off to 
expose unlabeled tissue.
Risks to patient treatment. The design of this study prevents undertreatment by concluding every 
procedure with standard of care FSA histology. Thus, complete tumor resection will always be 
confirmed using the standard method regardless of TPFM sensitivity. However, if TPFM 
demonstrates poor specificity, there is a risk that additional tissue could be removed that would not 
otherwise be (overtreatment). This is unlikely given our preliminary data in which BCC could be 
readily distinguished from healthy tissue and because the appearance of BCC is typically very 
apparent on both TPFM and FSA, but to further guard against this possibility, Dr. Ibrahim and Dr. 

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Sipprell will wait for FSA confirmation before further excisions unless they determine that BCC is both 
obvious on TPFM and the excision of additional tissue would not have a significant impact on the 
patient’s cosmetic outcome or reconstruction.   
Risks to privacy. In order to reduce the risk to patient privacy image data will be deidentified 
before removing it from the clinic.  PHI will be stored in RedCap and at RDS, where patient files 
would anyway be kept.  Thus, this study poses not significant increase in risk to the patient’s 
privacy.
Benefits. Positive margins may be more rapidly diagnosed for patients in the study, resulting in 
faster treatment and less time spent with open surgical wounds. At present, patients with positive 
margins for BCC must wait approximately 1 hour mid-surgery per stage of excision whereas TPFM 
imaging can be done in a few minutes. The development of new methods for real-time imaging of 
surgical pathology would have major impact on the health care by improving the efficiency of Mohs 
surgery. In comparison to the benefits, the risks are minimal since the investigation will maintain the 
standard of care for final margin confirmation and in case of any ambiguity in TPFM imaging. 
Consequently, the risk to benefit assessment is strongly in favor of benefit.
9. Costs for participation
There will be no additional costs to participate in this study beyond normal treatment costs. 
10. Payment for participation
There will be no payment for participation.
11. Subject Withdrawals
This is a single visit study with no follow up.  Thus subject withdrawal is unlikely, but could happen 
if a patient were unable to receive therapy during the clinic visit.  In this case, the subject would be 
withdrawn from the study and no data collected. 
12. Data Analysis:
The primary goal of this study is to evaluate the ability of TPFM to evaluate Mohs surgical margins 
during treatment of BCC. Consequently, analysis will compare the margin status as determined by 
TPFM to that determined on subsequent FSA.  Analysis will be performed for each stage of excision 
and lumped for all stages, but because not all surgeries have more than 1 stage, most likely analysis 
of stages 2 and higher will not be well-powered.  
Secondary goals will investigate the source of disagreements between FSA and TPFM, which are 
expected to include both limitations of FSA (poor tissue coverage, freezing artifacts, etc.) as well as 
TPFM (poor tissue flatness in the image plane, problems with staining or contrast, etc.).  This 
analysis will use images of FSA and TPFM images to determine the source of disagreement, 
providing both better insight into the overall sensitivity of the technique as well as limitations that 
could be addressed through technical improvements in microscope hardware.    

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A final analysis will look at excisions where TPFM enabled faster correction of positive margins.  
The time savings for patients will be measured, and the benefit to patients estimated.
13. Data and Safety Monitoring Plan
Data and Safety Monitoring Entities
The operating surgeon will provide safety monitoring during study activities.  The study PI will 
provide data and safety monitoring outside of the clinic.
Patient Safety Monitoring
Dr. Ibrahim will monitor imaging as part of treatment and report any suspected alterations of care to 
the study team and the RSRB.
Protection of patient privacy
All image data will be coded at the time of consent so as to protect identity of the patient. Consent 
forms and demographic information will be stored in the clinic.
Data Security
Research materials obtained from patients will include fluorescent images, digital copies of
histology slides and basic demographic information (age, sex, race) for enrollment. Each dataset will 
be deidentified and computerized using the protocol –assigned ID number in the URMC RedCap 
database. 
Storage of deidentified image data will occur at URMC using a secure server not connected to the 
internet. Deidentified and digital histological data will be shared with the clinical and research 
communities as appropriate, and we will invite the pathology community to evaluate or use 
deidentified image data as scientifically appropriate. PHI such as demographic data will be stored at 
URMC in RedCap.  
PI Oversight Plan
We have attached a separate PI oversight plan.
14. References:
1. 
M. G. Giacomelli, T. Yoshitake, L. C. Cahill, H. Vardeh, L. M. Quintana, B. E. Faulkner-
Jones, J. Brooker, J. L. Connolly, and J. G. Fujimoto, "Multiscale nonlinear microscopy and 
widefield white light imaging enables rapid histological imaging of surgical specimen 
margins," Biomed. Opt. Express 9, (2018).
2. 
L. C. Cahill, M. G. Giacomelli, T. Yoshitake, H. Vardeh, B. E. Faulkner-Jones, J. L. 
Connolly, C.-K. Sun, and J. G. Fujimoto, "Rapid virtual hematoxylin and eosin histology of 
breast tissue specimens using a compact fluorescence nonlinear microscope," Lab. Investig. 
00, 1–11 (2017).
3.  
T. Yoshitake, M. G. Giacomelli, L. M. Quintana, H. Vardeh, L. C. Cahill, B. E. Faulkner-
Jones, J. L. Connolly, D. Do, and J. G. Fujimoto, "Rapid histopathological imaging of skin 

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and breast cancer surgical specimens using immersion microscopy with ultraviolet surface 
excitation," Sci. Rep. 8, 4476 (2018).
4.
M. G. Giacomelli, B. E. Faulkner-Jones, L. C. Cahill, T. Yoshitake, D. Do, and J. G. 
Fujimoto, "Comparison of nonlinear microscopy and frozen section histology for imaging of 
Mohs surgical margins," Biomed. Opt. Express 10, 4249–4260 (2019).
5. 
K. N. Elfer, D. Tulman, S. J. Luethy, A. Sholl, J. Silberstein, and J. Q. Brown, "Virtual 
H&amp;E Whole-mount Fluorescence Histology of the Entire Prostate Surface for Real-time 
Surgical Guidance," in Biophotonics Congress: Biomedical Optics Congress 2018 
(Microscopy/Translational/Brain/OTS) (OSA, 2018), p. MF1A.4.
6. 
L. C. Cahill, J. G. Fujimoto, M. G. Giacomelli, T. Yoshitake, Y. Wu, D. I. Lin, H. Ye, O. M. 
Carrasco-Zevallos, A. A. Wagner, and S. Rosen, "Comparing histologic evaluation of 
prostate tissue using nonlinear microscopy and paraffin H&E: a pilot study.," Mod. Pathol. 
32, 1158–1167 (2019).
7. 
V. D. Ching-Roa, C. Z. Huang, S. F. Ibrahim, B. R. Smoller, and M. G. Giacomelli, "Real-
time Analysis of Skin Biopsy Specimens with 2-Photon Fluorescence Microscopy," JAMA 
Dermatology 158, 1175–1182 (2022).