Limits...
Improved tools for the Brainbow toolbox.

Cai D, Cohen KB, Luo T, Lichtman JW, Sanes JR - Nat. Methods (2013)

Bottom Line: In the transgenic multicolor labeling strategy called 'Brainbow', Cre-loxP recombination is used to create a stochastic choice of expression among fluorescent proteins, resulting in the indelible marking of mouse neurons with multiple distinct colors.Here we present several lines of mice that overcome limitations of the initial lines, and we report an adaptation of the method for use in adeno-associated viral vectors.We also provide technical advice about how best to image Brainbow-expressing tissue.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Brain Science, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
In the transgenic multicolor labeling strategy called 'Brainbow', Cre-loxP recombination is used to create a stochastic choice of expression among fluorescent proteins, resulting in the indelible marking of mouse neurons with multiple distinct colors. This method has been adapted to non-neuronal cells in mice and to neurons in fish and flies, but its full potential has yet to be realized in the mouse brain. Here we present several lines of mice that overcome limitations of the initial lines, and we report an adaptation of the method for use in adeno-associated viral vectors. We also provide technical advice about how best to image Brainbow-expressing tissue.

No MeSH data available.


Processing a Brainbow image(a) Original image. (b) Region boxed in a. (c) Deconvolution, resulting in decreased noise without sacrificing spatial resolution. (d) Intensity normalization, expanding perceptible color range. (e) Color shift correction (see also Supplemenatry Fig. 10). (f) Fully processed image.Yellow arrows indicate sequence of the image processing. White arrowheads indicate corresponding objects in the original and color-shift corrected images. Bars are 10µm in a and f, 3µm in b–e.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3713494&req=5

Figure 6: Processing a Brainbow image(a) Original image. (b) Region boxed in a. (c) Deconvolution, resulting in decreased noise without sacrificing spatial resolution. (d) Intensity normalization, expanding perceptible color range. (e) Color shift correction (see also Supplemenatry Fig. 10). (f) Fully processed image.Yellow arrows indicate sequence of the image processing. White arrowheads indicate corresponding objects in the original and color-shift corrected images. Bars are 10µm in a and f, 3µm in b–e.

Mentions: Brainbow images must be post-processed to maximize color information, but care is needed to avoid introducing artifacts. Often, one begins by reducing noise. Because confocal laser scanning of multicolor stacks is generally done at speeds of ~1 µs per pixel or less to save time, the small number of photons collected for each pixel gives rise to sufficient shot noise to cause perceptible local color differences. This problem can be minimized by slower scanning or averaging of multiple scans, but when this is infeasible, simple filtering and deconvolution methods are helpful (Fig. 6a–c). For example, median or Gaussian filters with 0.5–2 pixel radius reduces color noise, but at the expense of resolution. Deconvolution algorithms (see Online Methods) are more challenging to use than simple filters but can remove color noise without compromising spatial resolution (Supplementary Fig. 11).


Improved tools for the Brainbow toolbox.

Cai D, Cohen KB, Luo T, Lichtman JW, Sanes JR - Nat. Methods (2013)

Processing a Brainbow image(a) Original image. (b) Region boxed in a. (c) Deconvolution, resulting in decreased noise without sacrificing spatial resolution. (d) Intensity normalization, expanding perceptible color range. (e) Color shift correction (see also Supplemenatry Fig. 10). (f) Fully processed image.Yellow arrows indicate sequence of the image processing. White arrowheads indicate corresponding objects in the original and color-shift corrected images. Bars are 10µm in a and f, 3µm in b–e.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3713494&req=5

Figure 6: Processing a Brainbow image(a) Original image. (b) Region boxed in a. (c) Deconvolution, resulting in decreased noise without sacrificing spatial resolution. (d) Intensity normalization, expanding perceptible color range. (e) Color shift correction (see also Supplemenatry Fig. 10). (f) Fully processed image.Yellow arrows indicate sequence of the image processing. White arrowheads indicate corresponding objects in the original and color-shift corrected images. Bars are 10µm in a and f, 3µm in b–e.
Mentions: Brainbow images must be post-processed to maximize color information, but care is needed to avoid introducing artifacts. Often, one begins by reducing noise. Because confocal laser scanning of multicolor stacks is generally done at speeds of ~1 µs per pixel or less to save time, the small number of photons collected for each pixel gives rise to sufficient shot noise to cause perceptible local color differences. This problem can be minimized by slower scanning or averaging of multiple scans, but when this is infeasible, simple filtering and deconvolution methods are helpful (Fig. 6a–c). For example, median or Gaussian filters with 0.5–2 pixel radius reduces color noise, but at the expense of resolution. Deconvolution algorithms (see Online Methods) are more challenging to use than simple filters but can remove color noise without compromising spatial resolution (Supplementary Fig. 11).

Bottom Line: In the transgenic multicolor labeling strategy called 'Brainbow', Cre-loxP recombination is used to create a stochastic choice of expression among fluorescent proteins, resulting in the indelible marking of mouse neurons with multiple distinct colors.Here we present several lines of mice that overcome limitations of the initial lines, and we report an adaptation of the method for use in adeno-associated viral vectors.We also provide technical advice about how best to image Brainbow-expressing tissue.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Brain Science, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
In the transgenic multicolor labeling strategy called 'Brainbow', Cre-loxP recombination is used to create a stochastic choice of expression among fluorescent proteins, resulting in the indelible marking of mouse neurons with multiple distinct colors. This method has been adapted to non-neuronal cells in mice and to neurons in fish and flies, but its full potential has yet to be realized in the mouse brain. Here we present several lines of mice that overcome limitations of the initial lines, and we report an adaptation of the method for use in adeno-associated viral vectors. We also provide technical advice about how best to image Brainbow-expressing tissue.

No MeSH data available.