SLICE-compatible fluorescent dyes and proteins

SLICE excitation and emission filter wavelengths

SLICE is a light-sheet microscope designed for standardized, multicolor imaging of cleared tissue. Achieving optimal performance, however, requires careful selection of fluorescent dyes tailored to the system's excitation laser and emission filters. Here we outline fluorescence channel configurations and recommend compatible dyes to help you obtain the highest signal-to-background ratio and photostability for extended imaging sessions. SLICE supports three-color imaging using the following channels and excitation and emission wavelengths:

Channel Excitation (nm) Original Emission Filter (nm) New Emission Filter (nm)

Blue Narrow

455

513±11

513±11

Blue

455

-

510±40

Green

520

576±12

575±25

Red

640

680±15

700±38

Note that brain tissue is known to exhibit autofluorescence in the blue channel due to the presence of melanin, vitamin A1 derivatives, and lipofuscin. This can significantly reduce the signal-to-background ratio in the blue channel. Consider employing an autofluorescence quenching strategy during sample preparation to mitigate background fluorescence.

Fluorophore Choices

Organic dyes and fluorescent proteins that can be used for SLICE imaging are listed in the tables below, along with excitation, emission, and detection efficiencies which are defined as:

  • Excitation efficiency: The fraction that an incident photon at a given wavelength will be absorbed by the fluorophore.

  • Emission efficiency: The fraction of the dye’s emission spectrum that passes through the detection filter.

  • Detection efficiency: The fraction of incident excitation photons that ultimately produce detectable fluorescence photons within the detection filter bandwidth.

Organic Dyes

Organic dyes are generally preferred for imaging with the SLICE system due to their superior brightness, photostability, and resistance to photobleaching.

* Recommended dyes for SLICE imaging

Channel Dye Excitation Efficiency (%) Original Emission Efficiency (%) Original Detection Efficiency (%) New Emission Efficiency (%) New Detection Efficiency (%)
Blue Narrow Alexa 430 82 14 11 14 11
Alexa 488 18 36 6 36 6
Atto 465 99 25 25 25 25
Brilliant Violet 480 79 21 17 21 17
Cy2 33 32 11 32 11
Cyan 500 97 25 24 25 24
FITC 56 43 24 43 24
iFluor 450 99 34 34 34 34
NeuroTrace 500/525 35 23 8 23 8
Oregon Green 22 26 6 26 6
Pacific Green 36 27 10 27 10
SYTOX blue 66 15 10 15 10
YOYO-1 46 33 15 33 15
Blue Alexa 430 82 14 11 42 34
Alexa 488 18 36 6 76 14
Atto 465 99 25 25 73 72
Brilliant Violet 480 79 21 17 76 60
Cy2 33 32 11 71 23
Cyan 500 97 25 24 81 79
FITC 56 43 24 82 46
iFluor 450 99 34 34 84 83
NeuroTrace 500/525 35 23 8 57 20
Oregon Green 22 26 6 66 15
Pacific Green 36 27 10 79 28
SYTOX blue 66 15 10 70 46
YOYO-1 46 33 15 71 33
Green Alexa 514 99 18 18 45 45
Alexa 532 65 24 16 59 38
Alexa 546 31 44 14 72 22
Alexa 555* 51 34 17 63 32%
Alexa 568 23 11 3 29 7%
Cy3 51 42 21 69 35%
PE-Texas Red 55 7 4 20 11
SYTOX green 33 22 7 32 11
Red Alexa 633 83 22.5 19 42 35
Alexa 635 86 18 15 29 25
Alexa 647* 83 46 38 71 59
Alexa 660 77 45.4 35 87 67
Alexa 680 44 22 10 80 35
Cy5 84 47 39 69 58
eFluor 660 77 47 36 69 53

Fluorescent Proteins

While fluorescent proteins offer genetic specificity, their brightness and photostability often fall short of organic dyes.

Channel Protein Excitation Efficiency (%) Original Emission Efficiency (%) Original Detection Efficiency (%) New Emission Efficiency (%) New Detection Efficiency (%)
Blue Narrow ECFP 89 21 19 21 19
EGFP 56 43 24 43 24
mCerulean3 89 21 19 21 19
mNeonGreen 19 44 8 44 8
mTurquoise 92 20 18 20 18

Blue

ECFP

 89 21 19 79 70

EGFP

 56 43 24 84 47
mCerulean3 89 21 19 72 64
mNeonGreen 19 44 8 75 14
mTurquoise 92 20 18 69 63
Green

Citrine

87 13 11 30 26
DsRed 51 34 17 56 29
EYFP 77 14 11 31 24
FusionRed 29 12 3 28 8
mCherry 26 4 1 16 4
mRuby 38 14 5 29 11
RFP 41 24 10 45 18
tdTomato 60 30 18 50 30
Venus 81 13 11 28 27
Red APC 79 30 23 51 40
iRFP670 100 38 38 63 63
iRFP682 77 42 32 72 55