Fig. 1.
Methodology for detection of single RNA molecules by quantitative
FISH and digital imaging microscopy (13). Probes used are described
in (14). (A) The amount of light emitted per probe was calibrated
by measuring the TFI from a known number of probes in an imaged volume
(15). The TFI was plotted against the number of fluorochrome molecules
(five per probe) to generate the regression curve. The slope is equal
to the TFI per fluorochrome (curve a = CY3, curve b = CY5, curve c
= FITC). A normalized TFI per probe was used to calculate the number
of probes hybridized at discrete point sources of light in a restored
image (15). (B) The accuracy of the technique was tested by optically
sectioning and restoring immobilized CY3-labeled probes adsorbed to
glass cover slips. (C) The most frequent occurrences of TFI values
per CY3 probe plotted as a histogram are comparable to the normalized
value (solid bar) from the solution experiments (16). (D) One optical
plane of an NRK cell after in situ hybridization (17) with calibrated
CY3 probes to beta-actin mRNA shows a number of bright foci superimposed
on a diffuse background arising from out-of-focus light. Red arrow,
bead = 0.099 µm; yellow arrow, two transcription sites.
(E) The fluorescent probe distribution after image restoration with
an iterative constrained algorithm, EPR (18). The restored image reveals
discrete points of light, which allow accurate measurement of the
TFI emanating from each point source (bead is restored to a point
source). The image is a two-dimensional (2D) projection of the 3D
restored image. (F) The thresholded image (19) is rendered so that
the cell is viewed from the bottom looking upward into the nucleus
(20). The red surface portrays the boundary of the nuclear envelope.
The opening in the center of the nuclear surface is a result of truncating
the upper optical sections. (G) Enlargement of (F) showing the dimensions
of a restored transcription site in the nucleus (cross hairs), in
contrast to a 100-nm bead (yellow arrow), the most intense object
in the image, restored to a point source (21). (H) The integrated
TFI value of each point was mapped to a single voxel and the number
of probes hybridized assigned a color and frequency [1 = violet, 25.2%;
2 = blue, 33.6%; 3 = green, 17.5%; 4 = yellow, 11.6%; 5 = orange,
6.8%; 5 = red, 10 = white, <5%; n = 681 molecules per cell, 95%
efficiency of hybridization was estimated from a binomial distribution
(22)] (23). In (D) to (F), scale bar = 5 µm; in (B) and
(G), scale bar = 1 µm.
