Introduction to the Lumi-Imager
1. Introduction to the Lumi-Imager
The Lumi-Imager F1 Workstation (Cat. range of 1:10,000 (a hundredfold better
No. 2 012 847; Figure 24) allows rapid, than X-ray film) with precision and line-
quantitative analysis of chemilumines- arity (Figure 25).
cent or fluorescent signals without X-ray
Detection and Quantification with the Lumi-Imager
film. It can measure light over a dynamic
log
detected
intensities Lumi-Imager
X-Ray Film
0.01 0.1 1 10 100 log pg
Figure 25. Comparison of Linearity, Lumi-Imager
vs. X-ray Film. X-ray film gives a linear response to
signals over a 1:100 dynamic range. In contrast, Lumi-
Imager gives a linear response to signals over a
1:10,000 range.
Figure 24. The Lumi-Imager F1 Workstation. The
Lumi-Imager F1 Workstation can capture and quantify
chemiluminescent and fluorescent signals. Without
3 using X-ray film, it can analyze blots, gels, or microtiter
plates. It also produces visible light (pseudo-white
light transillumination or top illumination) to perform
analyses that rely on chromogenic signals. Pseudo-
white light transillumination also may be used to scan
signals on archived X-ray films.
The Workstation has two parts, a computer and the
light-detecting instrument. The computer uses Lumi-
Analyst software to control the instrument and analyze
results. The instrument is a mini-darkroom that houses
fixed-focus optics, a CCD camera (Charge-Coupled
Device camera, which converts incoming light into
quantifiable electrical signals), and a sample drawer.
150
Introduction to the Lumi-Imager
Applications for the Lumi-Imager
This chapter gives a brief overview of the Imager. Topics discussed in this chapter
capabilities and operation of the Lumi- include:
For information on this topic Turn to Starting on
Section page
Applications for the Lumi-Imager 1.1 151
Quantification with the Lumi-Imager 1.2 154
Detection and Quantification with the Lumi-Imager
Example of an Exposition and 1.3 155
Quantification Procedure
1.1 Applications for the Lumi-Imager
The Lumi-Imager is ideal for analyzing microplates, as well as many other appli-
DIG System signals on blots, gels, and cations (Table 1).
Table 1. Applications for Different Lumi-Imager Modes
Instrument Mode Applications Examples
Luminescence Analysis of chemilumines- ● Analysis of chemi-
cent signals from:
● Southern, Northern, or
Western blots up to
luminescent signals
produced by DIG-
labeled probe-target
3
24 x 30 cm hybrids
● Dot blots (Figures 3.3–3.5)
● Arrays ● Quantification and
● Colony or plaque lifts molecular weight
● Microplate assays, up to analysis of proteins on
4 plates western blots (e.g.
with the Lumi-Light Plus
Western Blotting Kit)
● Quantification of
microtiter plates assays
(e.g. reporter gene
assays)
Fluorescence/UV ● Direct analysis of ● Quantification of nucle-
Transillumination agarose or acrylamide ic acids on gels stained
gels stained with with ethidium bromide
fluorescent dyes or SYBR Green
● Quantification of ● Quantification of
microplates with UV proteins on gels
transparent bottoms stained with SYPRO
Red or Orange
151
Introduction to the Lumi-Imager
Applications for the Lumi-Imager
Instrument Mode Applications Examples
Top Illumination ● Imaging of any opaque ● Photograph of
object (petri dish, colonies or plaques on
photographs, etc.) petri dishes, for
● Densitometric analysis comparison with
of colored bands on plaque lifts
blots or in gels ● Quantification of
proteins on gels
stained with Coomassie
Detection and Quantification with the Lumi-Imager
Blue or silver stain
Pseudo-white Light ● Direct analysis of ● Quantification of
acrylamide gels stained proteins on acrylamide
with chromogenic dyes gels stained with
● Analysis of X-ray films Coomassie Blue or
silver stain
nylon membrane by the Southern blot method. A DIG-
labeled probe specific for the RFLP was then hybrid-
ized to the blot. Probe-target hybrids were detected in
a chemiluminescent reaction with CDP-Star and vis-
ualized with a 20 min exposure in the Lumi-Imager.
Data courtesy of H. van der Heide and F. R. Mooi,
RIVM Utrecht, The Netherlands.
Results and conclusion: Differences in restriction
pattern, indicative of the RFLP, are clearly visible in the
Figure 26. Analysis of an RFLP on a Southern Blot Bordetella pertussis samples after only a 20 min analy-
with the Lumi-Imager. Different strains of Bordetella sis of the blot. The Lumi-Imager could also be used to
3 pertussis were analyzed for presence of an RFLP
(restriction fragment length polymorphism). DNA sam-
ples from each strain were digested with restriction
easily quantify or determine the size of the restriction
fragments in the various strains (i.e., by comparing
them with DNA standards included on the blot).
enzymes, separated on a gel, and transferred to a
152
Introduction to the Lumi-Imager
Applications for the Lumi-Imager
Detection and Quantification with the Lumi-Imager
Figure 27. Quantification of high and low abun- Results and conclusion: The Lumi-Imager was able
dance transcripts on a Northern Blot with the to detect and quantify transcripts of the housekeeping
Lumi-Imager. Samples (3 µg) of mRNA from each of gene actin (left panel, lanes, 1– 4), an mRNA present in
four different types of tissue (liver HepG2, HeLa, nor- large amounts in the tissues. It could also detect and
mal human liver, and human muscle) were separated quantify transcripts of fibrinogen (left panel, lanes
electrophoretically, blotted onto a nylon membrane, 5– 8), an mRNA present in small amounts in the tis-
and hybridized with two DIG-labeled probes. The sues. The instrument could accurately quantify differ-
chemiluminescent signals from the probes (left panel) ences of mRNA content in the tissues for both the high
were detected with the Lumi-Imager (CDP-Star sub- (right panel, lanes 1– 4) and low abundance (right
strate, 2 min exposure). The results were quantified panel, lanes 5– 8) transcripts.
with the Lumi-Imager (right panel).
A B
3
Figure 28. Detection of Recombinant Bacterial were detected in the Lumi-Imager (B). An image of all
Colonies with the Lumi-Imager. Bacterial colonies the colonies on the plate (A) was also produced in the
were plated on an agar petri dish. The colonies were Lumi-Imager by photographing the plate under top
then analyzed for expression of a particular gene with illumination. Comparison of the two images makes it
a DIG-labeled probe. The chemiluminescent signals easy to detect colonies expressing the target gene.
(with CSPD substrate) from the hybridization probes
153
Introduction to the Lumi-Imager
Quantification with the Lumi-Imager
1.2 Quantification with the Lumi-Imager
Every aspect of the Lumi-Imager is de- balling” of complex blots, gels, or mic-
signed to allow rapid, accurate quantifica- roplates. For instance, preset functions
tion of chemiluminescent and fluorescent in the LumiAnalyst software can easily
signals. For example, the Lumi-Imager quantify relative abundance of signals
Detection and Quantification with the Lumi-Imager
offers these features to optimize and (Figure 27), determine mass/molecular
streamline the quantification of chemi- weight or analyze different colored sig-
luminescent and fluorescent signals: nals on the same blot or gel (Figure 30).
● A digitizing device (CCD) rather than A complete description of the capabilities
film for recording signals. of the Lumi-Imager is beyond the scope of
The CCD (Charge-Coupled Device) this article. For details on these features
camera offers higher resolution and and others, see our Lumi-Imager home
wider dynamic range than any film- page (http://biochem.roche.com/lumi-
based system. For instance, the CCD imager/index.htm) or contact your local
allows quantification of both strong Roche Molecular Biochemicals represen-
and weak bands on the same blot, tative.
Figure 29).
● Ability to display and analyze data in
many ways.
The sophisticated LumiAnalyst software
can eliminate the need for time-con-
suming manual measurement and “eye-
3
Figure 29. Accurate Analysis of Both Weak and The middle panel shows the result with an intermedi-
Strong Signals on the Same Blot. A Southern blot ate setting (240– 3000). The right panel shows the re-
containing widely variable amounts of restriction di- sult with a narrow setting (240-800).
gested DNA was hybridized to a DIG-labeled probe Result: The “ display range” feature of the Lumi-Imag-
that recognizes an RFLP. Hybridized restriction frag- er allows easy, accurate quantification of both weak
ments were detected with CDP-Star and visualized in and strong signals from a single captured image. Pro-
the Lumi-Imager (single 20 min exposure). After the ducing a comparable display with X-ray film would
original image was captured in the Lumi-Imager, the require three separate exposures, and the dynamic
“ Display Range” feature of the LumiAnalyst software range limitations of the film would prohibit accurate
was used to display the same image in three different quantification of the weak and strong signals on any
ways. The left panel was generated with the instru- single exposure.
ment set at the broadest dynamic range (270– 30 000).
154
Introduction to the Lumi-Imager
Example of an Exposition and Quantification Procedure
Detection and Quantification with the Lumi-Imager
Figure 30. Use of Two-color Analysis to Deter- and fluorescein green (right upper panel) fluorescence
mine Molecular weight of fluorescently-labeled were displayed simultaneously in the Lumi-Imager.
DNA. A single DNA was amplified and labeled with Result: The molecular weight of the green PCR prod-
fluorescein during PCR. The labeled product was uct (arrow, right panel) can be directly compared to
mixed with a Texas Red-labeled DNA molecular the red molecular weight standards in the same lane
weight marker and the mixture was separated on an (highlighted lane, left panel). This makes the molecular
agarose gel. Other lanes on the gel contained only the weight determination (left lower panel) much more
molecular weight marker. Texas Red (left upper panel) accurate.
3
1.3 Example of an Exposition and Quantification
Procedure
The procedure below summarizes how to Note: The procedure below is merely an
use the Lumi-Imager to display and quan- overview. For more details on Lumi-Im-
tify chemiluminescent signals on a blot. ager functions and settings to use, see the
As this procedure demonstrates, obtain- documentation for the LumiAnalyst soft-
ing quantitative results with the Lumi- ware. For an example of a typical blot
Imager is very simple; the LumiAnalyst quantification experiment, see Figure 27.
software does most of the work.
155
Introduction to the Lumi-Imager
Example of an Exposition and Quantification Procedure
Step Action
Start the computer and launch the LumiAnalyst program.
● Select “ Experiment” from the File menu.
● Enter the relevant data for your experiment.
Note: This information will help you retrieve the experimental data later
from the LumiAnalyst Image Database.
Click on “ LumiCapt” on the Main menu and capture an image of the blot by
Detection and Quantification with the Lumi-Imager
doing the following:
● Choose “ LumiAnalyst Capture” .
● Set “ Exposure Time” appropriate for the experiment.
Example: Set exposure time to 10 min if you are analyzing a typical
Southern blot with CDP-Star chemiluminescent alkaline phosphatase
substrate.
● Choose the “ Chemiluminescence” analysis mode.
● Place the sample (sealed bag containing blot and substrate) in the sample
drawer of the Lumi-Imager with the glowing side facing up.
Note: For details on setting up a chemiluminescent assay, see Chapter 2.
● Define a file name.
● Click the “ Start” button to capture an image of the chemiluminescent
signals on the blot.
Note: The image will be displayed automatically when the capture is
complete.
● Define an area of interest (“ AOI” ) on the captured image.
● Save the image of this AOI to a new file.
● Perform the rest of the procedure on this new, smaller image.
3 Select “ Edit” on the main menu and use “ Display Range” to adjust the display
scale of the image.
Note: The settings under “ Display Range” have no effect on the
quantification process or results.
Click on “ Show Tool Bar” , select the 1D-Gel Tool Bar, and do the following:
● Click the “ Lanes” button and select the lanes to be analyzed.
● Click the “ Find bands” button and select the band(s) to be analyzed in each
lane.
Note: The sensitivity of the “ Find Bands” function can be adjusted by
using the “ Minimum Band Height” setting.
● To adjust the pattern of bands displayed, do one or both of the following:
● Use “ Add Bands” to select bands that you want to measure, but that
were not automatically found.
● Use “ Delete Bands” to remove bands that you don’t want to measure.
Click on the “ Background” button and apply a background correction.
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Introduction to the Lumi-Imager
Example of an Exposition and Quantification Procedure
Step Action
Analyze the chosen lanes and bands by doing the following:
● Click on the “ Mass/BLU” button and choose “ Amount” or “ BLU” from the
Show menu that appears.
Note: BLU (Biochemical Light Units) are absolute measurement units for
expressing the area under each peak (i.e. signal resulting from a band).
● Click the “ Relative Abundance” button.
Result: The displayed data will show the relative intensity of each
Detection and Quantification with the Lumi-Imager
analyzed band as a function of parameters set with the “ Options” button.
(Other options for displayed data are “ Amounts” and “ Per cent” .)
● Select “ Calibrate” to choose a standard band (e.g., in the reference
standard), then specify the amount of substance in that band.
● Use the “ Update” option to recalculate the amounts of substance in each
lane relative to the standard.
Tip: For further analysis, you can export the calculated experimental data
to a spreadsheet program (such as Microsoft Excel) by selecting “ DDE to
Excel” from the File menu.
● Click the “ Save” button on the 1D-Gel Tool Bar.
● Click the “ Save new” button to save the experimental data to a new file in
the LumiAnalyst Image Database.
3
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