Laser-assisted Thermal-expansion Microinjection
Graduate School of Science, Nagoya University
Here we report a novel microinjection method designated as "Laser-assisted Thermal-expansion Microinjection" (patent application number (2004) 361448). This
method generate extremely high pressure so that we could use capillaries with fine tips around 0.1µm to inject materials. Thus, precise injection was possible, and
damage to cells and cell-components was drastically reduced. High pressure at the tip enabled us to inject plasmid DNAs into more than 50 plant cells by using a
single capillary, although injection into the plant cell (having cell wall) by using such a fine tip is impossible by conventional methods. Advantages and examples of
this simple method will be shown and discussed.
[Introduction] [Results & Discussion]
We previously succeeded in vitro pollen
tube guidance to the female gametophyte,
by using Torenia fournieri that has a naked
female gametophyte (Higashiyama et al.,
Plant Cell, 1998). Typical chemotropism is
observed in this system (Fig. 1, top panels).
We identified the “synergid cell” on the side
of egg cell as the source of the attractant,
by laser ablation experiments (Higashiyama
e t a l ., S ci e nce , 2 00 1 ; Fig. 1 , b o tt om
panels). Although chemical of the
a tt ra cta n t h a s b ee n u n kn o w n, i t w a s
suggested that the attractant might be a
molecule synthesized in the
synergid cell. We are now
tryin g to establish cDNA
library of the synergid cell.
We are planning RNAi
analysis using the EST data Fig.2 Diagram of the Laser-assisted Thermal-expansion Microinjection (LTM)
of it. In this study, we try to
develop novel injection Heat-induced expansion of liquid in a sealed capillary generate
system to introduce materials Fig.1 Pollen tube attraction by the synergid cell high pressure, which enables injection by capillaries with the tip-
i n t o t h e s y n e r g i d c e l l (A) to (D): A pollen tube chasing a manipulated female gametophyte. diameter around 0.1µm (Knoblauch et al., Nature Bitotech., 1999).
e f f i c i e n t l y, a l t h o u g h i t i s Bottom panels: laser ablation experiment showing that the synergid cell in As the heat source, we chose laser beam which enabled us to
d i f f i c u l t b y c o n v e n t i o n a l the female gametophyte is the source of the attractant. directly heat hydrophobic liquid containing laser absorber though
methods. the capillary glass and to precisely control the injection flow.
Instead of air pressure or oil pressure through silicone tube,
IR laser is irradiated to the capillary through optical fiber.
Injection flow can be precisely controlled by adjusting output
level of the laser power. To avoid back-filling of the
capillary, output level is gradually increased but not
decreased in a series of injection.
Fig.3 Equipments of LTM
The capillary holder is connected to Nd:YAG laser with optical fiber.
Due to high pressure (approximately 20 By using LTM, we could
10 atm per 1 ), it was possible to use readily inject materials
capillaries with the tip around 0.1µm. into the female gameto-
Fig. 4 shows comparison of capillary phytic cells and their
for conventional method and LTM. components of Torenia,
LTM capillaries were made by Sutter 10 as we aimed. LTM will
P97 puller. Such capillaries with fine allow us to perform
tips could not be used for conven- novel approach, inclu-
tional method (oil pressure) at all. 5
ding RNAi analysis in
However, we could use the capillaries the synergid cell by
for LTM without clogging the tip. More directly injecting double
than 5 0 toba cco BY-2 cells were thick tip fine tip fine tip
injected in a single capillary. method
Fig.4 Capillaries with fine tips around 0.1µm for LTM 10µm 20µm
SEM images of tips of conventional (~1.3µm; top on the left) and LTM (~0.1µm; bottom on the left)
capillaries and efficiency of injection with these capillaries (right) are shown. To examine the efficiency, Fig.5 LTM to the naked female gametophyte of Torenia
plasmids with Alexa-dextran 10k were injected into tobacco BY-2 cells, as in the case of Fig. 5, by using a Injection of plasmids with Alexa 546-dextran 10K into the central cell nucleus (left) and the
single capillary (up to 20 cells). synergid cell cytosol (right) is shown.
Nuclear injection drastically increases To test the ability of precise
the transformation rate than the case of injection by LTM, we further
cytosolic injection. However, nuclear tried more smaller objects.
injection in plant cells is difficult, and St r i k i n g l y, i n j e c t i o n o f
reports are still rare. We found that fluorochromes into yeasts was
onion epidermal cells were good system possible as shown in Fig. 7.
t o s e t u p t h e c o n d i t i o n o f n u cl e a r This suggests that LTM could
i n j e c t i o n u s i n g LT M , a l t h o u g h , b y be applied to a lot of kinds of
conventional methods, rigid cell wall cells, including prokaryotic
hamper the injection. As shown in Fig. 6, cells and organelle.
some population of injected cells were We are preparing for sell-
transformed and expressed GFP as ing products and looking for
tr a n s ie n t e xp r e s si o n . D e sp it e t h e co l l a b o r a t o r s t o t e s t LT M
successful injection and high viability of toward various types of cells
cells, transformation rate is still low and organelle. Please contact 10µm
(below 10%). Investigation of good us if you are interested in LTM
condition for plant transformation by 50µm (email@example.com).
microinjection is now on progress.
Fig.6 Transformation of onion epidermal cells by nuclear injection Fig.7 Injection into yeasts
Top panels: an epidermal cell just after nuclear injection (LTM) of plasmids with Alexa 546-dextran 10K. Bottom A diploid cell of yeast W303 at the log phase was hold by the aspiratory capillary (“left hand”
panels: a transformed epidermal cell 16 hours after injection, expressing NLS+GFP (bottom right). Arrows and as seen in the top panel) and was injected with Alexa 488-phalloidin using LTM.
arrow heads indicate nuclei and insertion marks of the capillary, respectively.