SLICE-compatible fluorescent dyes and proteins
SLICE excitation and emission filter wavelengths
SLICE is a high-throughput light-sheet microscope designed for standardized, multicolor imaging of cleared brain tissue. Achieving optimal performance, however, requires careful selection of fluorescent dyes tailored to the system's excitation laser and emission filter. 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 excitation and emission channels:
|
Excitation |
Emission filter |
|
|
Blue |
450 nm |
513±11 nm |
|
Green |
520 nm |
576±12 nm |
|
Red |
640 nm |
680±15 nm |
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
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 with high fluorescent efficiency.
Blue (450) channel, with Blue (513-22) Emission filter
| Dye |
EX Efficiency |
EM Efficiency |
|
Alexa 430 |
88% |
14% |
|
Alexa 488* |
14% |
36% |
Green (520) channel, with Green (576-23) Emission filter
| Dye |
EX Efficiency |
EM Efficiency |
|
Alexa 514 |
99% |
18% |
|
Alexa 532 |
65% |
24% |
|
Alexa 546 |
31% |
44% |
|
Alexa 555* |
51% |
34% |
Red (640) channel, with Red (680-30) Emission filter
| Dye |
EX Efficiency |
EM Efficiency |
|
Alexa 633 |
83% |
22.5% |
|
Alexa 635 |
86% |
18% |
|
Alexa 647* |
83% |
46% |
|
Alexa 660 |
77% |
45.4% |
Fluorescent Proteins
While fluorescent proteins offer genetic specificity, their brightness and photostability often fall short of organic dyes.
Blue (450) channel, with Blue (513-22) Emission filter
| Protein |
EX Efficiency |
EM Efficiency |
|
GFP |
49% |
44% |
|
NeuroTrace 500/525 |
27% |
23% |
Green (520) channel, with Green (576-23) Emission filter
| Protein |
EX Efficiency |
EM Efficiency |
|
tdTomato |
60% |
28% |
|
RFP |
41% |
23% |
|
mCherry |
26% |
13% |