Light Sources for Microscopy Focus on Fluorescence Microscopy Regan Baird, Ph.D. Focus on Fluorescence Microscopy Regan Baird, Ph.D.

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Presentation transcript:

Light Sources for Microscopy Focus on Fluorescence Microscopy Regan Baird, Ph.D. Focus on Fluorescence Microscopy Regan Baird, Ph.D.

Jablonski Diagram Fluorescence Spectra

White Light provides All Wavelengths?

Spectra of White Florescent Light

Spectrum of Light

Common Sources In Microscopy Natural Filament Arc Solid State

Illumination Needs Spectral Requirements Directed Intense Even Illumination

Natural sources of light Sunlight Fire Full Spectrum Difficult to Harness No Overnight Experiments Spectrum Depends On Fuel Difficult to Control Avoid Wind!

Filament Lamps

Halogen Lamp Spectra Typically Used for Brightfield Imaging Techniques

Arc Lamps

Mercury Metal Halide Xenon Peak levels are not comparable

Spectral Comparison 340nm and 380nm make Xenon ideal for Fura-2 imaging (Ca++)

Solid State Sources LED LED Hybrid Lasers

Light Emitting Diode Don’t be fooled by the peak heights

Light source Comparison

Filtered LED Source

Light Source Comparison

Common Fluorescence SOURCES SourceLifetimeStability Consumable Cost Wavelength Range Warm- up Shutter/S witch Speed Alignment Time to 50% degradatio n (hr) FlickerHazardHeatGreen Mercury100-$200UV minExternal+++Varies++++ NO Metal Halide 2000+$ minExternal NO Xenon1000++$ minExternal NO LED >1 min5kHZ YES Lasers UV-IR> 1 min1MHz Yes

Thoughts on Light Source Comparisons Use Compatible Filters!!! Where does the measurement matter most? What is the unit of measure? What is the area of illumination? What is the hour number on the source? What is the cost of ownership?