Research Techniques Made Simple: Confocal Microscopy AdaobiNwaneshiudu, MD PhD1, ChristianeKuschal, PhD,2 Fernanda H, Sakamoto, MD, PhD3, R. Rox Anderson, MD3, Kathryn Schwarzenberger, MD4, Roger C. Young, MD, PhD5 1The University of Chicago, Department of Medicine, Section of Dermatology, Chicago, IL 2National Institutes of Health, Bethesda MD 3 Wellman Center for Photomedicine, Department of Dermatology, Massachusetts General Hospital 4 Department of Medicine, Division of Dermatology, University of Vermont College of Medicine 5 Department of Obstetrics and Gynecology, University of Vermont College of Medicine

Confocal Microscopy Developed by Minsky in 1955 Optical imaging technique using point illumination via a pinhole to eliminate out of focus signal Pinhole is conjugate to the focal point of the objective lens of the microscope Advantages Increased optical resolution and contrast Facilitates 3-D reconstruction of sample Allows collection of serial optical sections from thick samples Allows surface profiling of samples, e.g., skin

Schematic Diagram of Confocal Microscopy Laser provides excitation light through focal point of objective lens to specimen for high intensity fluorescence Computer or CCD camera collects all “point images” & reconstructs the image High intensity fluorescence reflects off a dichroic mirror and hits 2 motorized mirrors Light is focused on pinhole and then measured by photomultiplier tube Motorized mirrors scan the laser across the specimen causing fluorescence Emitted fluorescence descanned by motorized mirrors and passes through dichroic mirror

Types of Confocal Microscopes Laser scanning confocal microscope – most widely used Spinning disk confocal microscope (Nipkow disk) Programmable array microscope Deconvoluted microscope – enables optical sectioning of the tissue Multi-photon imaging microscope In vivo reflectance confocal microscopes – enables surface profiling of thick tissues, like the skin

In vivo RCM in Melanoma Surveillance 1b 50mM 2 50mM 3 50mM Fig 1: (a) Dermoscopy image of an 8-mm-diameter lesion suspicious of melanoma with focal broadening of the pigment network, (b) In vivo RCM - Dark focus with regular bright-pigmented cells around dark dermal papillae at the dermo-epidermal junction. Diagnosis: lentiginous junctional nevus with mild dysplastic features. (Guitera et al, 2009) Fig 2: Numerous atypical cells (both dendritic and large roundish cells) at the dermoepidermal junction of a lentigo maligna of the cheek (Guitera et al, 2010) Fig 3: Atypical cobblestone pattern with small bright nucleated cells of the epidermis of a lentigo maligna of the cheek (Guitera et al, 2010)

Limitations of Confocal Microscopy Resolution limit due to signal to noise ratio, caused by a relative small number of detectable photons. Using more sensitive photo-detectors or increasing the intensity of the laser point source may increase the resolution limit Photobleaching of the fluorescent probe: Only a few minutes of illumination can alter the molecular structure of a fluorescent dye and can decrease the intensity of fluorescence significantly Phototoxicity of the fluorescent probe: When a fluorescent dye interacts with excitation light, photons are absorbed by the dye and viable cells can get damaged due to the photoreactive dye Chromatic and spherical aberration: two different fluorescent dyes can create a visual shift in an image which is a result of chromatic aberration of the lens used, causing an optical artifact that impair confocal image quality. Using perfluorodecalin as mounting medium can reduce such aberrations

LSCM versus SCEM Scanning confocal electron microscopy (SCEM) is another scanning technique where 3 dimensional images can be acquired The main difference: SCEM cannot be used to image living cells In SCEM, a focused electron beam illuminates the sample, and depth resolution is obtained by placing the collection optics symmetrically to the illumination optics The resolution is much higher compared to LSCM due to high energies of electrons (200keV) compared with photons (2eV)

LSCM versus SCEM Liang et al. (2006) used both SCEM and LSCM to characterize hair abnormalities in trichothiodystrophy (TTD) patients, who show fragile hair that breaks easily (due to decreased sulfur content). 1 2 Fig 4: Fig 5: 3 4 Fig 4: Confocal microscopy showing hair autofluorescence: left: trichoschisis, right: trichorrhexisnodosa- like fracture Fig 5: Scanning electron microscopy: (1) Grooved, irregular hair surface. (2) Flattened hair causing a ribbon-like appearance. (3) Trichorrhexisnodosa-like fracture. (4) Trichoschisis. The original magnification in mm is indicated by the length of the row of dots.

REFERENCES Guitera P, Pellacani G, Crotty KA, Scolyer RA, Li LX, Bassoli S, Vinceti M, Rabinovitz H, Longo C, Menzies SW (2010) The impact of in vivo reflectance confocal microscopy on the diagnostic accuracy of lentigomaligna and equivocal pigmented and nonpigmentedmacules of the face. J Invest Dermatol 130(8):2080-91 Guitera P, Pellacani G, Longo C, Seidenari S, Avramidis M, Menzies SW (2009) In vivo reflectance confocal microscopy enhances secondary evaluation of melanocytic lesions. J Invest Dermatol 129(1):131-8 Liang C, Morris A, Schlücker S et al. (2006) Structural and Molecular Hair Abnormalities in Trichothiodystrophy. J Invest Dermatol 126(10):2210.