FLUORESCENCE FILTERS FOR FISHFLUORESCENCE IN SITU HYBRIDIZATION

What You Need To Know For
Optimal Performance

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Many factors influence
the quality of FISH
preparations andthe images obtained
from them.

Specimen
Processingand storage

LightsourceMicroscope

ProbesFilters

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Fluorescence Filters
How do Fluorescence Filters affect the most important image
characteristics when viewing FISH samples so that you can:?
Score slides moreeasily?

Reduceuncertainty?

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Reduceeye strain?

Save time?

Increasethroughput?

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Fluorescence Filters
Filters dotwo things:
1TRANSMIT(allow to “pass” through)the desired wavelengthsof light (color), and thedesired amount(brightness) of lightAND

2

REJECT(block) undesired lightto a very high degree(high OD, or
Optical Density)

The combined effect is a filter with a very highsignal/noise ratio

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Fluorescence Filters
Two of these filters in a set work together to visualize fluorescence:

Selects andtransmits the optimalwavelengths of lightto “excite” a FISHprobe(s)+Selects andtransmits theoptimal wavelengthsof fluorescenceemitted by a FISHprobe(s)Excitation Filter

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By combining the right amount andcolor of excitation light with theright amount and color offluorescence emission, you canvisualize the desired FISH probe(s)Emission Filter

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Visualizing Fluorescence
Here’s How It Works:

Green Only

EMISSION FILTER:
blocks excitation light and transmits
the optimal wavelengths offluorescence emitted by a FISHprobe(s)VIOLET
BLUE
CYAN
GREEN
YELLOW
ORANGERED
DARK REDWhite Light

EXCITATION FILTER:
selects and transmits theoptimal wavelengths of light toexcite a FISH probe(s)CHROMA TECHNOLOGY CORP®

Some excitation lightreflects off sample;
dichroic rejects much ofthis but emission filter
must block what remains

Green/CyanCyan

DICHROIC MIRROR:
Reflects excitation lighttowards sample andtransmits longer
wavelengths, such as
fluorescence emission
towards eye or camera

Specimenlabeled with
Green FISHprobe

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Fluorescence Filters
By transmitting the most effective wavelengths of light, fluorescencefilters influence the 4 most important aspects of FISH images:

Contrast

Brightness

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Color
Separation

Registration(or alignment)www.chroma.com/FISHContrast
Fluorescence Signal vs. Background Noise
This is the Signal/Noise ratio of the image.

Problem:

Solution:

Low Contrast,
Difficult toscore slides

Maximize
Contrast

Low Signal/NoiseHigh Signal/Noise

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Benefit:Allow you to more easily see
the difference between real
signal and backgroundwww.chroma.com/FISHContrast

Good Contrast

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Inadequate Contrast

www.chroma.com/FISHContrast
A Taleof Two
Filter Sets

Good Contrast

Brighter(more signal to noise) – easy to score.

(more shaded area) but less contrast –more difficult to score.

AB

Overlap of filter set ‘A’ spectra with probe spectra(Grey-shaded area)

Excitation filter transmission spectra

Overlap of filter set ‘B’ spectra with probe spectra(Grey-shaded area)

Green FISH probe excitation and emission spectra

Emission filter transmission spectra

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www.chroma.com/FISHContrast
If Image ‘B’ is brighter, why is the contrast lower?

There is always background
fluorescence from various sources:
Cells and tissues themselves are autofluorescentFixatives and other reagents are autofluorescentBecause the noise
is higher…
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Fluorescent probes “stick” non-specificallywww.chroma.com/FISHContrast‘A’ has better Signal/Noise ratio = MORE CONTRAST

ABSignal

Noise(probe fluorescence)(background fluorescence)

++A

=B

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=‘B’ will detect
91% moresignal than ‘A’but…
‘B’ will detect
226% morenoise than ‘A’

www.chroma.com/FISHBrightness
Total intensity of fluorescence signal
Overall result of degree of excitation of sample (dichroic and excitation filter) plus level oftransmission of fluorescence (dichroic and emission filter)

Problem:

Solution:

Low Signal,
Difficult toscore slides

MaximizeSignal

Low Transmission

High Transmission

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Benefit:
Save time, increase throughput
and reduce eye strain withbrighter images

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Color Separation
Detect desired color(s) while rejecting undesired colors
Optimal filter combinations allow for best performance

Problem:

Solution:

Poor Color Separation
reduces certainty
and wastes timeMaximize
Color Purity

Poor Filter choiceAppropriate Filter choice

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}

Benefit:Distinguish between colors
more easily and exclude
unwanted colors

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Color Separation
Optimal color separation is not always simple
Considerations:
Requires appropriate combinations of probe colors
in specimens in order to reliably separate colors
Also dependent on quality of specimen processing
and balancing the degree of labeling of thedifferent probe colors
Often involves trade-offs between brightness andthe degree to which undesired colors are rejected
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Registration
Images are in register (aligned) with each other
Each filter set produces images that are aligned with images from other filter sets

Problem:

Solution:

Images offset fromeach other….whichcell is signal
coming from?

Align imagesby using
better filters

Poor filter materials

High Quality substrates

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Benefit:
Reduce uncertainty and savetime by allowing you to scorecells more easilywww.chroma.com/FISH

Registration
Filters are only one factor in image registration
Considerations:Factors such as the microscope filter turret are also
responsible for proper image registration
Image registered filters do not introduce any mismatch but
they cannot fix lack of registration caused by other sources
Typically only a factor when using a camera
Multiband sets for simultaneous viewing of multiple colors are
by definition “aligned with themselves”
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THANK YOU
Published in Bellows Falls, Vermont, US
Chroma optical filters and related products may be purchased online orvia phone. For more information, please e-mail sales@chroma.com,
visit www.chroma.com or call 800.824.7662.

CHROMA TECHNOLOGY CORP®

www.chroma.com/FISH