Confocal Microscopes. Evolution of Imaging

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1 Confocal Microscopes and Evolution of Imaging Judi Reilly Hans Richter Massachusetts Institute of Technology Environment, Health & Safety Office Radiation Protection

2 What is Confocal? Pinhole diaphragm in the Conjugated Focal plane = CONFOCAL Variable aperture Image by sathvi on withfriendship.com

3 History Principle of Confocal Microscopy patented by Marvin Minsky Out of focus areas are removed or suppressed Application in Material Sciences and Biology. Lasers used since end of 1980s Improvements Open beam confined use fiber optics enclosed systems Lasers new wavelengths physical large size to small size additional lasers Scanning technique mirrors spinning disc Photo detectors Marker dyes in biology Computer control and algorithms

4 Fluorescence Microscopy Material sciences: topographic imaging, photo-stimulated luminescence Biology Increase resolution Fluorescing structure, parts of cell, several parts at same time Dynamic processes in living cells movie of motion: growth & development of embryos, neural activities targeting agents: fluorescent markers The original jellyfish green fluorescent protein (GFP) Proteins Peptides RNA DNA lipids, lipoprotein drugs effects and pharmacokinetic behavior

5 Laser Scanning Machine VK-9700 Class II system 408 nm laser 0.9 mw possible to focus on a target with an uneven surface at high magnification. Surface projections and depressions can be measured without damaging the target area

7 2.5 watts water cooled Argon-Krypton laser 350 to 647 nm Fiber optic

10 Systems that contain a Class 3B or Class 4 Laser can be classed as a Class 1 system, if the accessible light poses no potential harm

11 Signage NOTICE: Eye and skin damage will occur for direct, momentary intrabeam exposure to Class 3B lasers. Post this WARNING SIGN at the entrance to the room and close the door whenever a Class 3B laser hazard exists. Class 3B LASER flip side Each registered laser user is responsible for: 1. Complying with all requirements of the MIT Laser Safety Program. 2. Wearing appropriate laser eyewear as necessary. 3. Conducting all laser activities in accordance with accepted good safety practices.

13 Laser beam is confined

17 A Few Examples of Fluorescent Protein Variants Protein (Acronym) Excitation Max Emission Max nm nm Green Fluorescent Protein GFP 395/ The original jellyfish green fluorescent protein (GFP) Emerald Azami Green Wasabi Blue Fluorescent Proteins EBFP Azurite Cyan Fluorescent Proteins ECFP Cerulean Yellow Fluorescent Proteins EYFP Topaz Orange Fluorescent Proteins Kusabira Orange dtomato Red Fluorescent Proteins mruby mstrawberry mraspberry

A Few Examples of Fluorescent Protein Variants Protein (Acronym) Excitation Max Emission Max nm nm Green Fluorescent Protein GFP 395/475 509 The original jellyfish green fluorescent protein (GFP)

18 A Few Examples of Fluorescent Protein Variants Protein (Acronym) Excitation Max Emission Max nm nm Green Fluorescent Protein GFP 395/ The original jellyfish green fluorescent protein (GFP) Emerald Azami Green Wasabi Blue Fluorescent Proteins EBFP Azurite Cyan Fluorescent Proteins ECFP Cerulean Yellow Fluorescent Proteins EYFP Topaz Orange Fluorescent Proteins Kusabira Orange dtomato Red Fluorescent Proteins mruby mstrawberry mraspberry

23 Two photon Microscopy Two photons absorbed, photons interact with the fluorophore at the same time and one photon emitted at a different wavelength. Provides a three dimensionality to image Longer wavelength more penetrating to tissue May image not only living cells, but tissue, embryos and animals Minimization of photobleaching and photodamage And improvements in noninvasive optical biopsy 1 photon excitation 2 photon excitation

24 Details of Two Photon Microscopy: Invented in 1990 by Watt Webb and Winfried Denk Fluorescent emission following two-photon excitation is exactly the same as emission generated in normal onephoton excitation High laser power is required to generate significant two-photon-excited fluorescence Achieved by focusing mode-locked (pulsed) lasers The power during the peak of the pulse is high enough to generate significant two-photon excitation while the average laser power remains fairly low.

25 Advantage: The narrow localization of two-photon excitation to the illumination focal point is the technique's advantage over confocal microscopy. Two-photon excitation only generates fluorescence at the focal plane, and since no background fluorescence is produced, a pinhole is not required.

26 Two types of ultrafast mode-locked laser systems are in use with current two-photon excitation microscopes: Ti:Sapphire laser (700 to 1100 nanometers) Nd:YLF laser (1047 nanometers)

28 Two Photon Microscope Beam Evaluation Open table optics Enclosed secured table optics

32 Imaging system on wheels used for animal research

33 LASER Hazard Assessment Visible & invisible laser light Standard Operating Procedure (SOP) Beam path contained in fiber optic or light pipe If NOT: potential to human exposure accessible to insertion of items or hand unnecessary items not in or near beam path are eyepieces usable when laser on dichroic filters used beam stop used and interlocked Laser eyewear available, appropriate, good condition 405, 458, 488, 514, 561, 594, 633 common wavelengths ask about use of UV or IR beams Training Is there a supervisor Are users trained Is there documentation

34 LASER Hazard Assessment Laser controlled by computer or human User log record (best practice as it is not required) Room is often used in dim light condition Maintenance done by vendor or by trained user Warning Signs & communications Secondary hazards: infectious material chemical hazards razor blades or other sharp objects animal use electrical (grounded, power strips, extension cords)

35 LASER Hazard Assessment Each setup different Is the user comfortable in answering your questions? Evaluate, ask questions, listen to what user is not saying Key is to get into the lab, review the setup, see how it is used evaluate potential hazards, communicate potential hazard reduce or mitigate hazard

38 Images can be seen on the Koch Institute web site Public Galleries

Future: Newer fluorophores fluorescence resonance energy transfer (FRET) fluorescence recovery after photobleaching (FRAP) fluorescence lifetime imaging microscopy (FLIM) More control of pulsed

39 Future: Newer fluorophores fluorescence resonance energy transfer (FRET) fluorescence recovery after photobleaching (FRAP) fluorescence lifetime imaging microscopy (FLIM) More control of pulsed lasers 3 photon techniques - deep tissue imaging Optogenetics - Neural activity and behavior Optical highlighters that are able to photoswitch (on or off) or Photoconvert to a new emission color upon stimulation with violet light Different techniques LSO to be aware of Changes/additions to initial setup New techniques Eyewear Communication

Special Techniques 1. Mark Scott FILM Facility

Special Techniques 1. Mark Scott FILM Facility Special Techniques 1 Mark Scott FILM Facility SPECIAL TECHNIQUES Multi-photon microscopy Second Harmonic Generation FRAP FRET FLIM In-vivo imaging TWO-PHOTON MICROSCOPY Alternative to confocal and deconvolution