Presentation on theme: "Research Techniques Made Simple: Confocal Microscopy"— Presentation transcript:
1 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, PhD51The University of Chicago, Department of Medicine, Section of Dermatology, Chicago, IL2National Institutes of Health, Bethesda MD3 Wellman Center for Photomedicine, Department of Dermatology, Massachusetts General Hospital4 Department of Medicine, Division of Dermatology, University of Vermont College of Medicine5 Department of Obstetrics and Gynecology, University of Vermont College of Medicine
2 Confocal Microscopy Developed by Minsky in 1955 Optical imaging technique using point illumination via a pinhole to eliminate out of focus signalPinhole is conjugate to the focal point of the objective lens of the microscopeAdvantagesIncreased optical resolution and contrastFacilitates 3-D reconstruction of sampleAllows collection of serial optical sections from thick samplesAllows surface profiling of samples, e.g., skin
3 Schematic Diagram of Confocal Microscopy Laser provides excitation light through focal point of objective lens to specimen for high intensity fluorescenceComputer or CCD camera collects all “point images” & reconstructs the imageHigh intensity fluorescence reflects off a dichroic mirror and hits 2 motorized mirrorsLight is focused on pinhole and then measured by photomultiplier tubeMotorized mirrors scan the laser across the specimen causing fluorescenceEmitted fluorescence descanned by motorized mirrors and passes through dichroic mirror
4 Types of Confocal Microscopes Laser scanning confocal microscope – most widely usedSpinning disk confocal microscope (Nipkow disk)Programmable array microscopeDeconvoluted microscope – enables optical sectioning of the tissueMulti-photon imaging microscopeIn vivo reflectance confocal microscopes – enables surface profiling of thick tissues, like the skin
5 In vivo RCM in Melanoma Surveillance 1b50mM250mM350mMFig 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)
6 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 limitPhotobleaching 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 significantlyPhototoxicity 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 dyeChromatic 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
7 LSCM versus SCEMScanning confocal electron microscopy (SCEM) is another scanning technique where 3 dimensional images can be acquiredThe main difference: SCEM cannot be used to image living cellsIn SCEM, a focused electron beam illuminates the sample, and depth resolution is obtained by placing the collection optics symmetrically to the illumination opticsThe resolution is much higher compared to LSCM due to high energies of electrons (200keV) compared with photons (2eV)
8 LSCM versus SCEMLiang 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).12Fig 4:Fig 5:34Fig 4: Confocal microscopy showing hair autofluorescence: left: trichoschisis, right: trichorrhexisnodosa- like fractureFig 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.
9 REFERENCESGuitera 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):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):131-8Liang C, Morris A, Schlücker S et al. (2006) Structural and Molecular Hair Abnormalities in Trichothiodystrophy. J Invest Dermatol 126(10):2210.